Anti-p53 antibodies

ABSTRACT

The invention relates to methods for producing an antibody which is specific for a mutant p53 polypeptide over wildtype p53 polypeptide, comprising using the mutant p53 polypeptide as an immunogen wherein the polypeptide comprises: (i) an antigen sequence, comprising an amino acid sequence of the mutant p53 polypeptide including the mutation and at least one amino acid immediately adjacent to the mutation, and (ii) a scaffold sequence for providing the antigen sequence in a solvent-accessible configuration, in particular wherein the scaffold sequence is thioredoxin. In the specific embodiments, antibodies against mutant p53 comprising R175H, R248Q or R273H are generated. Also disclosed are the uses of the antibodies for diagnosis, prognosis and stratification of patient groups and further encompasses the use of antibodies for imaging and treatment of cancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Apr. 12, 2019, is named2008187_0149_SL.txt and is 159,434 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies directed against mutant p53polypeptides and methods for producing the same.

BACKGROUND TO THE INVENTION

Evolutionary processes have resulted in the perfection of the immuneresponse, in order for highly specific recognition events that can besurmounted to combat evading almost all types of exogenous antigens(Flajnik and Kasahara, Nat Rev Gene (2010) 11(1):47-59). In vertebrates,both T- and B-lymphocytes have developed receptors that undergoantigen-induced selective rearrangements to detect the slightest ofvariabilities that may occur in highly related antigenic proteins fromsimilar species (Cooper and Alder, Cell (2006) 124(4):815-822), therebyestablishing a barrier against foreign invasion. This innate ability hasbeen exploited to generate a large number of antibodies against highlysimilar proteins with subtle structural variations (Lo et al., 2014Microbiol. Spectr. 2, AID—0007-2012). Moreover, antibodies have alsobeen made to detect subtle post-translational modifications such asphosphorylation, ubiquitination and acetylation on target amino-acidresidues, which has led to the mechanistic understanding of a widevariety of physiological and pathological processes. However, detectionof single amino acid changes, often noted due to missense mutations inthe cancer genomes, represent a significant challenge to the generationof selective antibodies, due to the high homology of the flankingregions on the parent proteins. Whilst attempts have been made to makemutation-specific antibodies that do not cross-react with the unmutatednative protein, most efforts have been thus far been unsuccessful(Bondgaard et al., Modern Pathology (2014) 27:1590-1598).

While mAbs against surface antigens such as EGFR, PD-1, etc. have beensuccessfully used in the clinical setting for treatment (Martinelli etal., 2009 Clinical and Experimental Immunology 158(1): 1-9; Philips andAtkins 2014, Int Immunol 27(1): 39-46), mAbs against nuclear antigensgenerally face a challenge in getting past the cell membrane to beeffective. However, recent studies have shown that antibodies againstnuclear antigens can indeed be therapeutically effective (Hong and Zeng,2012 Cancer Res 72(11): 3715), providing promise that the approach maybe successful in the future.

p53 is the most mutated gene across all cancer types, with over 50% ofall tumors exhibiting some form of genetic alterations in it (Forbes etal. 2015 Nucleic Acids Res 43, D805-D811.). Most mutations in p53 arefound in the central DNA-binding domain (DBD) that spans around 200amino-acids, and account for about 90% of all p53 mutations identified.Almost all DBD mutations lead to the loss of wild-type p53 functions,which are essential for tumor suppression and effective response totherapy, due to p53's multi-functional role in guarding the genome(Vousden and Lu, Nat Rev Cancer (2002) 2(8):594-604; Freed-Pastor andPrives, Genes Dev (2012) 26(12):1268-1286). Besides loss of wild-typefunctions, mutations in p53 also leads to two major phenomena that aidthe development of cancers and also inhibit effective response againsttherapy. One is the dominant-negative (DN) effect that occurs when amutant allele of p53 co-exists with the wild-type allele, therebyforming hetero-oligomers that result in the attenuation of the remainingwild-type p53 function (Kern et al., Science (1992) 256:827-830). Inaddition, the presence of mutant p53 allele alone, especially during thelater stages of cancers when the wild-type p53 allele is lost due toloss-of-heterozygozity, results in cancer cells becoming addicted to themutant p53 protein for survival, due to the gain of novel oncogenicfunctions (GOF) by mutant p53 that promotes survival and invasiveproperties (Sabapathy, Front Oncol (2015) 5:276). A causal role formutant p53 in these processes has been demonstrated by reducing itsexpression or inactivating its function, where both the DN and GOFfunctions can be rescued, allowing cancer cells to become more sensitiveto therapeutic treatment (Lee et al. Cancer Cell (2012) 22:751-764).While these data opens up the exciting possibility of targeting mutantp53, several challenge for successful inactivation of mutant p53 exist.Most importantly, it is to be noted that while all mutations in p53unequivocally lead to loss of its wild-type activities and in most casesthe DN effect, the GOF property appears to be selective for some mutantsand not all (Lee et al. Cancer Cell (2012) 22:751-764; Hanel et al.,Cell Death Diff (2013) 20:898-909), highlighting that targeting mutantp53 needs a more sophisticated approach to selectively detect andinactivate each individual p53 mutant.

Understanding the biology of mutant p53 has been fundamental touncovering mutant p53's role in carcinogenesis and response to therapy.While significant progress has been made in our knowledge of how mutantp53 exerts its pro-survival functions, there are limitations. Forexample, not all mutations are functionally similar (Sabapathy, 2015Front. Oncol. 5: 276), and the mutations found in the transactivationand oligomerization domains of p53—which are quite common in certainpopulations (Achatz et al., 2007 Cancer Lett. 245:96-102)—requirefurther evaluation.

Current technologies have utilized knock-in mouse models or human tumorcell lines that express only the mutant protein without the wildtypeprotein, which may not entirely reflect the role of the mutant proteinin co-existence with the wildtype protein.

Attempts to generate antibodies against mutant p53 have been made overthe decades, and monoclonal antibodies (mAb) against the mutant (PAb240)or the wild-type (PAb246) conformation of p53 are available (Gannon etal., EMBO J (1990) 9(5):1595-1602). However, these antibodies are notsufficiently specific and exhibit cross reactivity, and require veryselective conditions for the detection of the correct conformations thathave thus limited their use.

Vojtesek and Lane, J Cell Sci (1993), 105(3):607-612 describes anantibody designated DO-1 against the amino-terminal region of human p53,which shows a high level of specificity for human p53 without anycross-reactivity with mouse p53.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for producingan antibody which is specific for a mutant p53 polypeptide over wildtypep53 polypeptide, comprising using as an immunogen a peptide orpolypeptide comprising: (i) an antigen sequence, comprising an aminoacid sequence of the mutant p53 polypeptide including the mutation andat least one amino acid immediately adjacent to the mutation, and (ii) ascaffold sequence for providing the antigen sequence in asolvent-accessible configuration.

In some embodiments, the scaffold sequence is derived from a peptide orpolypeptide comprising a solvent-accessible sequence, and wherein theantigen sequence is inserted in, or substituted for all or part of, thesolvent-accessible sequence of the peptide or polypeptide. In someembodiments, the peptide or polypeptide comprising a solvent-accessiblesequence is a thioredoxin, and wherein the solvent-accessible sequenceis the active site sequence of the thioredoxin. In some embodiments, thepeptide or polypeptide used as an immunogen additionally comprises oneor more linker sequences between the antigen sequence and the scaffoldsequence. In some embodiments, the peptide or polypeptide used as animmunogen comprises at least two amino acid sequences of the mutant p53polypeptide. In some embodiments, the peptide or polypeptide used as animmunogen additionally comprises linker sequences between the at leasttwo amino acid sequences of the mutant p53 polypeptide. In someembodiments, the at least two amino acid sequences of the mutant p53polypeptide are non-identical. In some embodiments, the amino acidsequence of the mutant p53 polypeptide comprises at least 5 amino acids.In some embodiments, the mutant p53 polypeptide comprises a mutation inthe DNA-binding domain (DBD). In some embodiments, the mutant p53polypeptide comprises a mutation selected from one of R175H, R248Q,R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y, H179R,Y220C, and R337H. In some embodiments, the mutant p53 polypeptidecomprises, or consists of, the amino acid sequence of one of SEQ ID NOs:3 to 16.

In another aspect, the present invention provides an antibody, orantigen binding fragment, obtained or obtainable by a method forproducing an antibody according to the invention.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is specific for a mutant p53 polypeptideover wildtype p53 polypeptide.

In some embodiments, the mutant p53 polypeptide comprises a mutation inthe DNA-binding domain (DBD). In some embodiments, the mutant p53polypeptide comprises a mutation selected from one of R175H, R248Q,R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y, H179R,Y220C and R337H. In some embodiments, the mutant p53 polypeptidecomprises, or consists of, the amino acid sequence of one of SEQ ID NOs:3 to 16.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R175H p53polypeptide, optionally isolated, having the amino acid sequences i) tovi):

i) LC-CDR1: (SEQ ID NO: 31) QSLLNSGNQKSX₁; ii) LC-CDR2: (SEQ ID NO: 20)GAS; iii) LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT; iv) HC-CDR1:(SEQ ID NO: 32) GX₂TFTEYT; v) HC-CDR2: (SEQ ID NO: 33) IX₃PX₄X₅GX₆T;vi) HC-CDR3: (SEQ ID NO: 27) ARWGGDYV;

or a variant thereof in which one or two or three amino acids in one ormore of the sequences i) to vi) are replaced with another amino acid,where X₁=Y or N; X₂=F or Y; X₃=D or N; X₄=N or Y; X₅=N or S; and X₆=V orG.

In some embodiments, LC-CDR1 is one of QSLLNSGNQKSY (SEQ ID NO:19) orQSLLNSGNQKSN (SEQ ID NO:23). In some embodiments, HC-CDR1 is one ofGFTFTEYT (SEQ ID NO:25) or GYTFTEYT (SEQ ID NO:29). In some embodiments,HC-CDR2 is one of IDPNNGVT (SEQ ID NO:26) or INPYSGGT (SEQ ID NO:30).

In some embodiments, the antibody, or antigen binding fragment, has atleast one light chain variable region incorporating the following CDRs:

LC-CDR1: (SEQ ID NO: 31) QSLLNSGNQKSX₁; LC-CDR2: (SEQ ID NO: 20) GAS;LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT;

where X₁=Y or N.

In some embodiments, the antibody, or antigen binding fragment has atleast one light chain variable region incorporating the following CDRs:

LC-CDR1: (SEQ ID NO: 19) QSLLNSGNQKSY; LC-CDR2: (SEQ ID NO: 20) GAS;LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT.

In some embodiments, the antibody, or antigen binding fragment has atleast one light chain variable region incorporating the following CDRs:

LC-CDR1: (SEQ ID NO: 23) QSLLNSGNQKSN; LC-CDR2: (SEQ ID NO: 20) GAS;LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT.

In some embodiments, the antibody, or antigen binding fragment has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 32) GX₂TFTEYT; HC-CDR2: (SEQ ID NO: 33)IX₃PX₄X₅GX₆T; HC-CDR3: (SEQ ID NO: 27) ARWGGDYV;

where X₂=F or Y; X₃=D or N; X₄=N or Y; X₅=N or S; and X₆=V or G.

In some embodiments, the antibody, or antigen binding fragment has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 25) GFTFTEYT; HC-CDR2: (SEQ ID NO: 26) IDPNNGVT;HC-CDR3: (SEQ ID NO: 27) ARWGGDYV.

In some embodiments, the antibody, or antigen binding fragment has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 29) GYTFTEYT; HC-CDR2: (SEQ ID NO: 30) INPYSGGT;HC-CDR3: (SEQ ID NO: 27) ARWGGDYV.

In another aspect, the present invention provides an isolated lightchain variable region polypeptide, comprising the following thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 31) QSLLNSGNQKSX₁; LC-CDR2: (SEQ ID NO: 20) GAS;LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT;

where X₁=Y or N.

In some embodiments, LC-CDR1 is one of QSLLNSGNQKSY (SEQ ID NO:19) orQSLLNSGNQKSN (SEQ ID NO:23).

In another aspect, the present invention provides an isolated heavychain variable region polypeptide, comprising the following thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 32) GX₂TFTEYT; HC-CDR2: (SEQ ID NO: 33)IX₃PX₄X₅GX₆T; HC-CDR3: (SEQ ID NO: 27) ARWGGDYV;

where X₂=F or Y; X₃=D or N; X₄=N or Y; X₅=N or S; and X₆=V or G.

In some embodiments, HC-CDR1 is one of GFTFTEYT (SEQ ID NO:25) orGYTFTEYT (SEQ ID NO:29). In some embodiments, HC-CDR2 is one of IDPNNGVT(SEQ ID NO:26) or INPYSGGT (SEQ ID NO:30).

In another aspect, the present invention provides an isolated lightchain variable region polypeptide as described herein in combinationwith a heavy chain variable region polypeptide as described herein.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R175H p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

-   -   the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at        least 85% overall sequence identity to LC-CDR1: one of        QSLLNSGNQKSX₁ (SEQ ID NO:31), QSLLNSGNQKSY (SEQ ID NO:19) or        QSLLNSGNQKSN (SEQ ID NO:23); LC-CDR2: GAS (SEQ ID NO:20);        LC-CDR3: QNDHSYPLT (SEQ ID NO:21); and the heavy chain comprises        a HC-CDR1, HC-CDR2, HC-CDR3, having at least 85% overall        sequence identity to HC-CDR1: one of GX₂TFTEYT (SEQ ID NO:32),        GFTFTEYT (SEQ ID NO:25) or GYTFTEYT (SEQ ID NO:29); HC-CDR2: one        of IX₃PX₄X₅GX₆T (SEQ ID NO:33), IDPNNGVT (SEQ ID NO:26) or        INPYSGGT (SEQ ID NO:30); HC-CDR3: ARWGGDYV (SEQ ID NO:27); where        X₁=Y or N; X₂=F or Y; X₃=D or N; X₄=N or Y; X₅=N or S; and X₆=V        or G.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R175H p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

-   -   the light chain sequence has at least 85% sequence identity to        the light chain sequence of SEQ ID NO:18 or 22 (FIG. 24 ), or        SEQ ID NO:237 or 94 (FIG. 49 ), and;    -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of SEQ ID NO:24 or 28 (FIG. 25 ), or        SEQ ID NO:239, 240 or 241 (FIG. 50 ).

In another aspect, the present invention provides an antibody, orantigen binding fragment, optionally isolated, which is capable ofbinding to R175H p53 polypeptide, which is a bispecific antibody or abispecific antigen binding fragment comprising (i) an antigen bindingfragment or polypeptide as described herein, and (ii) an antigen bindingfragment capable of binding to a polypeptide other than R175H p53polypeptide.

In another aspect, the present invention provides an in vitro complex,optionally isolated, comprising an antibody, antigen binding fragment,or polypeptide as described herein bound to R175H p53 polypeptide or afragment thereof.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R248Q p53polypeptide, optionally isolated, having the amino acid sequences i) tovi):

i) LC-CDR1: (SEQ ID NO: 41) QSLLYSDGKTY; ii) LC-CDR2: (SEQ ID NO: 42)LVS; iii) LC-CDR3: (SEQ ID NO: 43) WQGTHFPLT; iv) HC-CDR1:(SEQ ID NO: 46) GYTFTDYY; v) HC-CDR2: (SEQ ID NO: 52) IX₇PKNGGT;vi) HC-CDR3: (SEQ ID NO: 53) AKX₈GGX₉DDY;

or a variant thereof in which one or two or three amino acids in one ormore of the sequences i) to vi) are replaced with another amino acid,where X₇=H or D; X₈=M or Q; and X₉=Y or F.

In some embodiments, HC-CDR2 is one of IHPKNGGT (SEQ ID NO:47) orIDPKNGGT (SEQ ID NO:50). In some embodiments, HC-CDR3 is one ofAKMGGYDDY (SEQ ID NO:48) or AKQGGFDDY (SEQ ID NO:51).

In some embodiments, the antibody, or antigen binding fragment, has atleast one light chain variable region incorporating the following CDRs:

LC-CDR1: (SEQ ID NO: 41) QSLLYSDGKTY LC-CDR2: (SEQ ID NO: 42) LVSLC-CDR3: (SEQ ID NO: 43) WQGTHFPLT.

In some embodiments, the antibody, or antigen binding fragment, has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 46) GYTFTDYY; HC-CDR2: (SEQ ID NO: 52) IX₇PKNGGT;HC-CDR3: (SEQ ID NO: 53) AKX₈GGX₉DDY;

where X₇=H or D; X₈=M or Q; and X₉=Y or F.

In some embodiments, the antibody, or antigen binding fragment, has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 46) GYTFTDYY; HC-CDR2: (SEQ ID NO: 47) IHPKNGGT;HC-CDR3: (SEQ ID NO: 48) AKMGGYDDY.

In some embodiments, the antibody, or antigen binding fragment, has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 46) GYTFTDYY; HC-CDR2: (SEQ ID NO: 50) IDPKNGGT;HC-CDR3: (SEQ ID NO: 51) AKQGGFDDY.

In another aspect, the present invention provides an isolated lightchain variable region polypeptide, comprising the following thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 41) QSLLYSDGKTY LC-CDR2: (SEQ ID NO: 42) LVSLC-CDR3: (SEQ ID NO: 43) WQGTHFPLT.

In another aspect, the present invention provides an isolated heavychain variable region polypeptide, comprising the following thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 46) GYTFTDYY; HC-CDR2: (SEQ ID NO: 52) IX₇PKNGGT;HC-CDR3: (SEQ ID NO: 53) AKX₈GGX₉DDY;

where X₇=H or D; X₈=M or Q; and X₉=Y or F.

In some embodiments, HC-CDR2 is one of IHPKNGGT (SEQ ID NO:47) orIDPKNGGT (SEQ ID NO:50). In some embodiments, HC-CDR3 is one ofAKMGGYDDY (SEQ ID NO:48) or AKQGGFDDY (SEQ ID NO:51).

In another aspect, the present invention provides an isolated lightchain variable region polypeptide as described herein in combinationwith a heavy chain variable region polypeptide as described herein.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R248Q p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

-   -   the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at        least 85% overall sequence identity to LC-CDR1: QSLLYSDGKTY (SEQ        ID NO:41); LC-CDR2: LVS (SEQ ID NO:42); LC-CDR3: WQGTHFPLT (SEQ        ID NO:43); and the heavy chain comprises a HC-CDR1, HC-CDR2,        HC-CDR3, having at least 85% overall sequence identity to        HC-CDR1: GYTFTDYY(SEQ ID NO:46); HC-CDR2: one of IX₇PKNGGT (SEQ        ID NO:52), IHPKNGGT (SEQ ID NO:47) or IDPKNGGT (SEQ ID NO:50);        HC-CDR3: one of AKX₈GGX₉DDY (SEQ ID NO:53), AKMGGYDDY (SEQ ID        NO:48) or AKQGGFDDY(SEQ ID NO:51); where X₇=H or D; X₈=M or Q;        and X₉=Y or F.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R248Q p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

-   -   the light chain sequence has at least 85% sequence identity to        the light chain sequence of SEQ ID NO:40 or 44 (FIG. 28 ), or        SEQ ID NO:251 or 252 (FIG. 57 ), and;    -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of SEQ ID NO:45 or 49 (FIG. 29 ), or        SEQ ID NO:253 or 254 (FIG. 58 ).

In another aspect, the present invention provides an antibody, orantigen binding fragment, optionally isolated, which is capable ofbinding to R248Q p53 polypeptide, which is a bispecific antibody or abispecific antigen binding fragment comprising (i) an antigen bindingfragment or polypeptide as described herein, and (ii) an antigen bindingfragment capable of binding to a polypeptide other than R248Q p53polypeptide.

In another aspect, the present invention provides an in vitro complex,optionally isolated, comprising an antibody, antigen binding fragment,or polypeptide as described herein bound to R248Q p53 polypeptide or afragment thereof.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R273H p53polypeptide, optionally isolated, having the amino acid sequences i) tovi):

i) LC-CDR1: (SEQ ID NO: 59) QSIVHNNGDTY; ii) LC-CDR2: (SEQ ID NO: 60)KVS; iii) LC-CDR3: (SEQ ID NO: 61) FQGSHLPLT; iv) HC-CDR1:(SEQ ID NO: 63) GFSFSDYY; v) HC-CDR2: (SEQ ID NO: 64) ISVGGTYT;vi) HC-CDR3: (SEQ ID NO: 65) VRDGNDGKFLG;

or a variant thereof in which one or two or three amino acids in one ormore of the sequences i) to vi) are replaced with another amino acid.

In some embodiments, the antibody, or antigen binding fragment, has atleast one light chain variable region incorporating the following CDRs:

LC-CDR1: (SEQ ID NO: 59) QSIVHNNGDTY LC-CDR2: (SEQ ID NO: 60) KVSLC-CDR3: (SEQ ID NO: 61) FQGSHLPLT.

In some embodiments, the antibody, or antigen binding fragment, has atleast one heavy chain variable region incorporating the following CDRs:

HC-CDR1: (SEQ ID NO: 63) GFSFSDYY HC-CDR2: (SEQ ID NO: 64) ISVGGTYTHC-CDR3: (SEQ ID NO: 65) VRDGNDGKFLG.

In another aspect, the present invention provides an isolated lightchain variable region polypeptide, comprising the following thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 59) QSIVHNNGDTY LC-CDR2: (SEQ ID NO: 60) KVSLC-CDR3: (SEQ ID NO: 61) FQGSHLPLT.

In another aspect, the present invention provides an isolated heavychain variable region polypeptide, comprising the following thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 63) GFSFSDYY HC-CDR2: (SEQ ID NO: 64) ISVGGTYTHC-CDR3: (SEQ ID NO: 65) VRDGNDGKFLG.

In another aspect, the present invention provides an isolated lightchain variable region polypeptide as described herein in combinationwith a heavy chain variable region polypeptide as described herein.

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R273H p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at least85% overall sequence identity to LC-CDR1: QSIVHNNGDTY (SEQ ID NO:59);LC-CDR2: KVS (SEQ ID NO:60); LC-CDR3: FQGSHLPLT (SEQ ID NO:61); and theheavy chain comprises a HC-CDR1, HC-CDR2, HC-CDR3, having at least 85%overall sequence identity to HC-CDR1: GFSFSDYY (SEQ ID NO:63); HC-CDR2:ISVGGTYT (SEQ ID NO:64); HC-CDR3: VRDGNDGKFLG (SEQ ID NO:65).

In another aspect, the present invention provides an antibody, orantigen binding fragment, which is capable of binding to R273H p53polypeptide, comprising a light chain and a heavy chain variable regionsequence, wherein:

-   -   the light chain sequence has at least 85% sequence identity to        the light chain sequence of SEQ ID NO:58 (FIG. 32 ) or SEQ ID        NO:247 (FIG. 53 ), and;    -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of SEQ ID NO:62 (FIG. 33 ) or SEQ ID        NO:248 (FIG. 54 ).

In another aspect, the present invention provides an antibody, orantigen binding fragment, optionally isolated, which is capable ofbinding to R273H p53 polypeptide, which is a bispecific antibody or abispecific antigen binding fragment comprising (i) an antigen bindingfragment or polypeptide as described herein, and (ii) an antigen bindingfragment capable of binding to a polypeptide other than R273H p53polypeptide.

In another aspect, the present invention provides an in vitro complex,optionally isolated, comprising an antibody, antigen binding fragment,or polypeptide as described herein bound to R273H p53 polypeptide or afragment thereof.

In another aspect, the present invention provides an antibody, antigenbinding fragment, or polypeptide as described herein conjugated to adrug moiety or a detectable moiety.

In another aspect, the present invention provides a chimeric antigenreceptor (CAR) comprising an antigen binding fragment or polypeptide asdescribed herein.

In another aspect, the present invention provides a cell comprising achimeric antigen receptor (CAR) as described herein.

In another aspect, the present invention provides a compositioncomprising the antibody, antigen binding fragment, polypeptide,conjugate, chimeric antigen receptor (CAR) or cell as described hereinand at least one pharmaceutically-acceptable carrier.

In another aspect, the present invention provides an isolated nucleicacid encoding the antibody, antigen binding fragment, polypeptide,conjugate, or chimeric antigen receptor (CAR) as described herein.

In another aspect, the present invention provides a vector comprisingthe nucleic acid as described herein.

In another aspect, the present invention provides a host cell comprisingthe vector as described herein.

In another aspect, the present invention provides a method for making anantibody, antigen binding fragment, polypeptide, conjugate, or chimericantigen receptor (CAR) as described herein, comprising culturing thehost cell under conditions suitable for the expression of a vectorencoding the antibody, antigen binding fragment, polypeptide orconjugate, and recovering the antibody, antigen binding fragment,polypeptide or conjugate

In another aspect, the present invention provides an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein for use in therapy, or in a method ofmedical treatment.

In another aspect, the present invention provides an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein in the manufacture of a medicament foruse in a method of medical treatment.

In another aspect, the present invention provides a method of treating adisease, the method comprising administering an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein to a patient suffering from a disease.

In another aspect, the present invention provides an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein for use in the treatment or preventionof cancer.

In another aspect, the present invention provides an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein in the manufacture of a medicament foruse in the treatment or prevention of cancer.

In another aspect, the present invention provides a method of treatingcancer comprising administering an antibody, antigen binding fragment,polypeptide, conjugate, chimeric antigen receptor (CAR) or cell asdescribed herein to a patient suffering from a cancer.

In another aspect, the present invention provides a method of preventingcancer or preventing a recurrence of cancer, the method comprisingadministering an antibody, antigen binding fragment, polypeptide,conjugate, chimeric antigen receptor (CAR) or cell as described hereinto a patient considered to be at risk of cancer or at risk of recurrenceof cancer.

In another aspect, the present invention provides a method, optionallyan in vitro method, comprising contacting a sample containing, orsuspected to contain, a mutant p53 polypeptide with an antibody, antigenbinding fragment, polypeptide, conjugate, chimeric antigen receptor(CAR) or cell as described herein and detecting the formation of acomplex of the antibody, antigen binding fragment, polypeptide,conjugate, CAR or cell with the mutant p53 polypeptide.

In another aspect, the present invention provides a method of diagnosinga disease or condition in a subject, the method comprising contacting,in vitro, a sample from the subject with an antibody, antigen bindingfragment, polypeptide, conjugate, chimeric antigen receptor (CAR) orcell as described herein and detecting the formation of a complex of theantibody, antigen binding fragment, polypeptide, conjugate, CAR or cellwith the mutant p53 polypeptide.

In another aspect, the present invention provides a method of selectingor stratifying a subject for treatment with a mutant p53polypeptide-targeted agent, the method comprising contacting, in vitro,a sample from the subject with an antibody, antigen binding fragment,polypeptide, conjugate, chimeric antigen receptor (CAR) or cell asdescribed herein and detecting the formation of a complex of theantibody, antigen binding fragment, polypeptide, conjugate, CAR or cellwith the mutant p53 polypeptide.

In another aspect, the present invention provides the use of anantibody, antigen binding fragment, polypeptide, conjugate, chimericantigen receptor (CAR) or cell as described herein for the detection ofa mutant p53 polypeptide in vitro or in vivo.

In another aspect, the present invention provides the use of anantibody, antigen binding fragment, polypeptide, conjugate, chimericantigen receptor (CAR) or cell as described herein as an in vitro or invivo diagnostic or prognostic agent.

In another aspect, the present invention provides the use of anantibody, antigen binding fragment, polypeptide, conjugate, chimericantigen receptor (CAR) or cell as described herein in a method fordetecting, localizing or imaging a cancer in vivo.

In another aspect, the present invention provides a method fordetecting, localizing or imaging a cancer in vivo, comprisingadministering an antibody, antigen binding fragment, polypeptide,conjugate, chimeric antigen receptor (CAR) or cell as described hereinto a subject, and detecting the antibody, antigen binding fragment,polypeptide, conjugate, chimeric antigen receptor (CAR) or cell.

In another aspect, the present invention provides a vaccine, optionallya cancer vaccine, comprising an immunogen as described herein.

In another aspect, the present invention provides an immunogen asdescribed herein for use in a method of vaccination, optionally againsta cancer.

In another aspect, the present invention provides the use of animmunogen as described herein in the manufacture of a medicament for usein the vaccination of a subject, optionally against a cancer.

In another aspect, the present invention provides a method forvaccinating a subject, optionally against a cancer, comprisingadministering to the subject an immunogen as described herein, therebyvaccinating the subject against a cancer.

In some embodiments in accordance with various aspects of the presentinvention, the cancer is a cancer comprising a cell or cells expressinga mutant p53 polypeptide, or comprising nucleic acid encoding a mutantp53 polypeptide. The mutant p53 polypeptide may correspond to theselected antibody, antigen binding fragment, polypeptide, conjugate,chimeric antigen receptor (CAR), cell or immunogen.

DESCRIPTION

Mutant p53 Polypeptides and Wildtype p53 Polypeptides

The p53 tumour suppressor is a protein which is encoded in humans by theTP53 gene. p53 plays a crucial role in cellular response to DNA damageand other genomic aberrations.

Activation of p53 leads to cell cycle arrest, DNA repair or apoptosis.Mutations of the gene encoding p53 have been implicated in thedevelopment and progression of a variety of cancers (Muller and Vousden,Cancer Cell (2014) 25:304-317).

Certain p53 mutations, including the hotspot mutations (R175H, R248W andR273H), not only result in the loss of p53-dependent tumor suppressoractivity, but also result in the acquisition of oncogenic activity.

A wildtype p53 polypeptide as referred to herein may comprise or consistof a reference amino acid sequence for p53 for a given animal. Referenceamino acid sequences for p53 can be retrieved from databases known tothe person skilled in the art. Such databases include GenBank, EMBL,DDBJ, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, ProteinData Bank, Ensembl and InterPro.

The animal may be a mammal. In some embodiments, the animal may be ahuman. In some embodiments, the animal may be a non-human mammal (e.g.rabbit, guinea pig, rat, mouse or other rodent (including any animal inthe order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows,e.g. dairy cows, or any animal in the order Bos), horse (including anyanimal in the order Equidae), donkey, and non-human primate).

In some embodiments a wildtype p53 polypeptide may comprise or consistof the amino acid sequence of UniProtKB-P04637 (P53_HUMAN):

(SEQ ID NO: 1) MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDS D

A mutant p53 polypeptide is a polypeptide having an amino acid sequencewhich is different to the amino acid sequence of the wildtype p53polypeptide, e.g. as a result of insertion, deletion or substitution ofone or more amino acids of the wildtype p53 polypeptide amino acidsequence.

Mutant p53 polypeptides may occur as a result of genetic mutation of theDNA encoding a p53 polypeptide causing a change in the amino acidsequence of the polypeptide. Mutations of p53 are known in the art, andare described for example in Muller and Vousden, Cancer Cell (2014)25:304-317, which is hereby incorporated by reference in itsentirety—see e.g. Table 1 spanning pages 305-308.

The present invention is concerned in particular with antibodies whichare capable of binding to mutant p53 polypeptides which are only verysubtly different from wildtype p53 polypeptide, and which do not exhibitsignificant binding to wildtype p53 polypeptide.

In some embodiments, the antibodies are capable of binding to a mutantp53 polypeptide which differs from wildtype p53 polypeptide by 10 aminoacids or fewer, e.g. 5, 4, 3 or 2 amino acids or fewer. In someembodiments, an antibody is capable of binding to a mutant p53polypeptide which differs from wildtype p53 polypeptide by one aminoacid.

That is, the present invention provides antibodies capable ofrecognising single amino acid mutant variants of p53.

In some embodiments, the amino acid(s) of the mutant p53 polypeptidewhich differ from the amino acid sequence of wildtype p53 are in thesequence of the DNA-binding domain.

The DNA-binding domain of p53 is evolutionarily conserved, and theskilled person is able to identify the DNA-binding domain for a givenwildtype p53 polypeptide by reference to the DNA-binding domain sequenceof e.g. human wildtype p53 polypeptide. The DNA-binding domain for humanwildtype p53 polypeptide corresponds to positions 101-306 of SEQ ID NO:1(see e.g. Freed-Pastor and Prives, Genes and Development (2012)26:1268-1286), having the following sequence:

(SEQ ID NO: 2) KTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPH HELPPGSTKR

Nine hot spots for mutation of p53 within the DNA binding domain of p53have been identified, corresponding to positions 248, 273, 175, 176,179, 220, 245, 249, 282 numbered relative to SEQ ID NO:1, and a furtherhotspot has been identified at position 337. In some embodiments, themutant p53 polypeptide to which an antibody according to the presentinvention specifically binds comprises an amino acid difference relativeto the wildtype p53 sequence at one or more of positions 248, 273, 175,176, 179, 220, 245, 249, 282 and 337. In some embodiments, the mutantp53 polypeptide comprises an amino acid difference relative to thewildtype p53 sequence at one of position 248, 273, 175, 176, 179, 220,245, 249, 282, or 337.

In some embodiments, an antibody according to the invention is capableof binding to a specific, known mutant p53 polypeptide. In someembodiments an antibody according to the present invention may becapable of binding to a p53 polypeptide comprising one of the followingamino acid substitutions numbered relative to SEQ ID NO:1: R175H, R2480,R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y, H179R,Y220C, R337H.

In some embodiments, an antibody according to the invention may becapable of binding to mutant p53 polypeptide comprising or consisting ofone of the following sequences:

Sequence (mutated residue relative to SEQ ID Mutationwildtype p53 shown bold, underlined) NO: R175HMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 3VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVR H CPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R248QMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 4VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNS SCMGGMN QRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R273HMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 5VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEV H VCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R248WMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 6VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNS SCMGGMN WRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD G245SMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 7VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNS SCMG SMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R273CMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 8VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEV C VCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R282WMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 9VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRD W RTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R249SMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 10VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNS SCMGGMNR SPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD G245DMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 11VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGDMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD C176FMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 12VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRR F PHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD H179YMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 13VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPH Y ERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD H179RMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 14VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPH R ERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD Y220CMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 15VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVP C EPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD R337HMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD SEQ IDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSS NO: 16VPSQKTYQGSYGFRLGFLHSGTAKSVICTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRE H FEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFK TEGPDSD

The skilled person is readily able to determine equivalent mutations ofthe p53 polypeptide in the amino acid sequence of p53 polypeptides ofother species, e.g. by sequence alignment.

Reference to a given p53 mutation numbered according to the amino acidsequence of the human wildtype p53 polypeptide (SEQ ID NO:1) includesequivalent mutations in homologous proteins to p53 in other species,e.g. mouse or rat.

For example, reference to “R175H” herein encompasses the equivalentarginine to histidine mutation at position 172 of the amino acidsequence for the mouse homologue of p53 (i.e. R172H)—see e.g. Olive etal, 2004 Cell 119, 847-860, which is hereby incorporated by reference inits entirety. Similarly, reference to “R175H p53 polypeptide” hereinencompasses the polypeptide comprising the equivalent R172H mutantpolypeptide of the mouse homologue of p53

Immunogens

The antibodies according to the present invention are produced accordingto methods described herein, raised using immunogens.

In the present invention, an immunogen is a peptide or polypeptidemolecule. As used herein, a “peptide” is a chain of two or more aminoacid monomers linked by peptide bonds. A peptide typically has a lengthin the region of about 2 to 50 amino acids. A “polypeptide” is a polymerchain of two or more peptides. Polypeptides typically have a lengthgreater than about 50 amino acids.

In the methods of the present invention antibodies are raised by amethod using as an immunogen a peptide or polypeptide comprising: (i) anantigen sequence, comprising an amino acid sequence of the mutant p53polypeptide including the mutation and at least one amino acid eitherside of the mutation, and (ii) a scaffold sequence for providing theantigen sequence in a solvent-accessible configuration.

Antigen Sequence

The antigen sequence comprises, or consists of, an amino acid sequenceof the mutant p53 polypeptide to which the antibody produced by themethod is capable of binding.

The amino acid sequence of the mutant p53 polypeptide of the antigensequence comprises the mutation; that is, the antigen sequence includesthe mutated position of the mutant p53 polypeptide relative to the aminoacid sequence of the wildtype p53 polypeptide.

The amino acid sequence of the mutant p53 polypeptide of the antigensequence additionally comprises at least one amino acid immediatelyadjacent to the mutation. That is, the sequence includes at least oneamino acid residue immediately upstream of (i.e. N-terminal to) orimmediately downstream of (i.e. C-terminal to) the mutated position inthe amino acid sequence of the mutated p53 polypeptide. The mutation isthereby presented in the context of the immediate sequence of aminoacids in which it occurs in the mutant p53 polypeptide.

By way of illustration, with reference to R175H mutant p53 representedby SEQ ID NO:1 above, the amino acid sequence of the mutant p53polypeptide comprises at least “RH”, or “HC”.

In some embodiments, the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise one of at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids immediately upstream of themutated position. In some embodiments, the amino acid sequence of themutant p53 polypeptide of the antigen sequence may comprise one of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids immediatelydownstream of the mutated position. In some embodiments, the amino acidsequence of the mutant p53 polypeptide of the antigen sequence maycomprise one of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsimmediately upstream of the mutated position, and one of at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids immediately upstream of themutated position.

In some embodiments, the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise or consist of one ofnot more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 12, 11, or 10amino acids immediately upstream of the mutated position. In someembodiments, the amino acid sequence of the mutant p53 polypeptide ofthe antigen sequence may comprise or consist of one of not more than 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 12, 11, or 10 amino acidsimmediately downstream of the mutated position. In some embodiments, theamino acid sequence of the mutant p53 polypeptide of the antigensequence may comprise or consist of one of not more than 25, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 12, 11, or 10 amino acids immediatelyupstream of the mutated position, and one of not more than 25, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 12, 11, or 10 amino acids immediatelyupstream of the mutated position.

In some embodiments, the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise or consist of one of1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14,1-15, or 1-20 amino acids immediately upstream of the mutated position.In some embodiments, the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise or consist of one of1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14,1-15, or 1-20 amino acids immediately downstream of the mutatedposition. In some embodiments, the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise or consist of one of1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14,1-15, or 1-20 amino acids immediately upstream of the mutated positionand one of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-13, 1-14, 1-15, or 1-20 amino acids immediately downstream of themutated position.

In some embodiments, the amino the amino acid sequence of the mutant p53polypeptide of the antigen sequence may comprise or consist of one of7-11, 3-7, or 2-5 amino acids immediately upstream and downstream of themutated position.

The amino acid sequence of the mutant p53 polypeptide (including themutation and upstream/downstream amino acids) may comprise or consist ofone of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, or 30 amino acids. In some embodiments, the amino acidsequence of the mutant p53 polypeptide may comprise or consist of notmore than 50, 45, 40, 35, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,12, 11, or 10 amino acids. In some embodiments, the amino acid sequenceof the mutant p53 polypeptide may be 2-50, 5-40, 5-30, 5-25, 5-20, 5-15,8-30, 8-25, 8-20, 8-15, 10-30, 10-25, 10-20, or 10-15 amino acids inlength. In some preferred embodiments the amino acid sequence of themutant p53 polypeptide may be 5-20 or 8-20 amino acids in length.

In some embodiments, the antigen sequence of the immunogen comprisesmore than a single copy of the mutation. That is, in some embodiments,the antigen sequence comprises two or more amino acid sequences of themutant p53 polypeptide. In embodiments wherein the antigen sequencecomprises multiple amino acid sequences of the mutant p53 polypeptide,each amino acid sequence of the mutant p53 polypeptide may independentlybe defined according to an embodiment described herein.

By way of example, with reference to R175H mutant p53 represented by SEQID NO:1 above, an antigen sequence comprising more than one amino acidsequence of the R175H mutant p53 polypeptide may comprise the sequence“QHMTEVVRHCPHHERCSDgsgTEVVRHCPHHER” (SEQ ID NO:90) (lower case indicatesa flexible serine/glycine linker sequence). It will be clear that thissequence comprises two amino acid sequences of the mutant p53polypeptide; (1) QHMTEVVRHCPHHERCSD and (2) TEVVRHCPHHER.

In some embodiments, the antigen sequence comprises one of 1, 2, 3, 4, 5or 6 amino acid sequences of the mutant p53 polypeptide. In someembodiments, the antigen sequence comprises at least 2, 3 or 4 aminoacid sequences of the mutant p53 polypeptide. In some embodiments, theantigen sequence comprises not more than 6, 5, 4 or 3 amino acidsequences of the mutant p53 polypeptide. In some embodiments, theantigen sequence comprises or consists of 2 or 3 amino acid sequences ofthe mutant p53 polypeptide.

In embodiments where the antigen sequence comprises more than one aminoacid sequence of the mutant p53 polypeptide, each amino acid sequence ofthe mutant p53 polypeptide may be identical. In some embodiments, anantigen sequence may comprise non-identical amino acid sequences of themutant p53 polypeptide. In some embodiments, each amino acid sequence ofthe mutant p53 polypeptide may be non-identical.

In some embodiments wherein the antigen sequence comprises more than oneamino acid sequence of the mutant p53 polypeptide, the antigen sequenceadditionally comprises linker sequence(s) between amino acid sequencesof the mutant p53 polypeptide. Linker sequences are describedhereinbelow.

Scaffold Sequence

The immunogen also comprises a scaffold sequence for providing theantigen sequence in a solvent-accessible configuration.

By providing the antigen sequence in a solvent-accessible configuration,the scaffold sequence provides the opportunity to raise antibodiesdirected against the antigen sequence of the immunogen. Specifically,such configuration allows antibody to physically contact the antigensequence.

Whether a scaffold sequence provides the antigen sequence in a solventaccessible configuration can be determined or predicted based on theamino acid sequence of the immunogen.

Solvent accessible and/or inaccessible amino acids or sequences of aminoacids may be predicted based on the sequence of amino acids, or based onthe three-dimensional structure of an amino acid sequence. Solventaccessibility can, for example, be predicted by calculating theaccessible surface area (ASA), as reviewed in Ali et al., Currentprotein and Peptide Science, 2014, 15(3), which is hereby incorporatedby reference in its entirety. ASA may be calculated, for example, usingthe Shrake-Rupley (rolling probe) algorithm, Z-layer integration,intersection, linear combinations of pairwise overlaps (LCPO), or powerdiagram.

Tools for predicting ASA for an amino acid sequence include artificialneural networks (ANN), support vector machines (SVM), and the Markovchain model (MCM). Software which can be used to predict solventaccessibility includes ASAview, ACCpro, PDBePISA, CCP4, GETAREA, DSSP,ProtSA, NACCESS, ACCESS, POPS-R, SERF, NetSurfP, ASAP, SANN and SABLE.ACCPro5.1 implements a 1 D-recursive neural network algorithm.

In some embodiments, the scaffold sequence is derived from a peptide orpolypeptide comprising a solvent-accessible sequence.

In some embodiments, the antigen sequence is provided in a solventaccessible configuration by being inserted in, or substituted for all orpart of, the solvent accessible sequence of the peptide or polypeptide.It will be appreciated that in such embodiments, the scaffold sequenceis interrupted by the antigen sequence.

In some embodiments, the antigen sequence may be inserted between twoamino acids within a solvent-accessible sequence of the peptide orpolypeptide from which the scaffold sequence is derived. In someembodiments, the antigen sequence may be inserted in place of one ormore residues of the solvent accessible sequence.

Solvent accessible and/or inaccessible amino acids or sequences of aminoacids are known or predicted for some peptides/polypeptides, and can beidentified by the skilled person with reference to databases known tothe skilled person, including ASAview. Solvent accessible and/orinaccessible amino acids or sequences of amino acids may also bepredicted based on the sequence of amino acids for apeptide/polypeptide, or based on the three-dimensional structure of anamino acid sequence for a peptide/polypeptide, using the tools describedhereinabove.

Any suitable scaffold sequence may be used in the immunogen according tothe present invention. In some embodiments, the scaffold sequence may beselected such that:

-   -   a) the antigen sequence inserted in the scaffold sequence is        presented on the surface of the folded scaffold sequence; and/or    -   b) the folding and/or the three-dimensional structure of the        scaffold sequence is not substantially changed or disrupted by        the antigen sequence; and/or    -   c) the scaffold sequence provides for high expression and/or        easy purification of the immunogen; and/or    -   d) the scaffold sequence is immunogenic.

The skilled person is able to readily identify scaffold sequencespossessing the above recited properties, e.g. by analysis ofscaffold/immunogen sequences using algorithm-based predictions fromamino acid sequences/protein structures, reference to databases, orempirical analysis.

In some embodiments, the peptide/polypeptide comprising asolvent-accessible sequence from which the scaffold sequence is derivedis a thioredoxin. Thioredoxin structure and function is described inCollet and Messens, Antioxid Redox Signal (2010) 13:1205-1216, which ishereby incorporated by reference in its entirety. Barrell et al. ProteinExpr Purif (2004) 33(1): 153-159 (hereby incorporated by reference inentirety) describes insertion of an amino acid sequence into the activesite of a bacterial thioredoxin for the production of antibodiesspecific to the inserted amino acid sequence.

In some embodiments the thioredoxin comprises the amino acid sequenceaccording to SEQ ID NO:17, or an fragment thereof having at least 20amino acids (optionally one of at least 30, 40, 50, 60, 70, 80, 90 or100 amino acids), having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (e.g.following alignment), and having the active site motif Cys-Gly-Pro-Cys(SEQ ID NO:91):

(SEQ ID NO: 17) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKG QLKEFLDANLA

In some embodiments, the solvent-accessible sequence of the thioredoxinis the active site sequence. The active site sequence of thioredoxincorresponds to the motif Cys-Gly-Pro-Cys, which is conserved inthioredoxins of diverse origin. In some embodiments, the antigensequence is inserted into, or substituted for all or part of, thissequence. In some embodiments the antigen sequence is inserted after theN-terminal Cys residue of the active site sequence, or after the Glyresidue, or after the Pro residue. In some embodiments, the antigensequence is inserted immediately upstream of the N-terminal Cys residueof the active site sequence in the amino acid sequence of thethioredoxin. In some embodiments, the antigen sequence is insertedimmediately downstream of the C-terminal Cys residue of the active sitesequence in the amino acid sequence of the thioredoxin.

In some embodiments the scaffold sequence comprises, or consists of, SEQID NO:17. In some embodiments the scaffold sequence comprises, orconsists of, an amino acid sequence having at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO:17, or a fragment thereof having at least 20 amino acids(optionally one of at least 30, 40, 50, 60, 70, 80, 90 or 100 aminoacids).

In some embodiments, the scaffold sequence may comprise one or moreadditional amino acids to facilitate surface orientation and/or solventexposure of the antigen sequence of the immunogen. In some embodiments,the scaffold may comprise amino acid residues at one of each end of theantigen sequence to facilitate surface orientation and/or solventexposure of the antigen sequence provided in the sequence of theimmunogen.

By way of illustration, with reference to the R175H immunogen shown inFIG. 1A (i.e. SEQ ID NO: 80), the scaffold (positions 1 to 35 andpositions 91 to 165 of SEQ ID NO:80) of the immunogen comprises anadditional proline residue adjacent to the antigen sequence insert, sothat the antigen sequence is flanked in the sequence of the immunogen byproline residues (positions 35 and 91 of SEQ ID NO:80).

Linker Sequence

The immunogen may comprise one or more linker sequences between aminoacid sequences. As explained above, the immunogen may comprise linkersequences between amino acid sequences of a mutant p53 polypeptidewithin the antigen sequence.

In some embodiments, the immunogen may additionally comprise one or morelinker sequences between the antigen sequence and scaffold sequence. Inembodiments wherein the antigen sequence is inserted in, or substitutedfor all or part of, a scaffold sequence, a linker sequence may beprovided at one or both ‘ends’ of the antigen sequence. By way ofillustration, the immunogen may comprise the following structure:

-   -   [scaffold sequence]-{linker}-[antigen        sequence]-{linker}-[scaffold sequence]

Linker sequences are known to the skilled person, and are described, forexample in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369,which is hereby incorporated by reference in its entirety.

In some embodiments, a linker sequence may be a flexible linkersequence. Advantageously, flexible linker sequences allow for relativemovement of the amino acid sequences which are linked by the linkersequence, enhancing accessibility of the sequence to antigen bindingmolecules. Flexible linkers are known to the skilled person, and severalare identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, incorporated by reference hereinabove. Flexible linkersequences often comprise high proportions of glycine and/or serineresidues.

In some embodiments, the linker sequence comprises at least one glycineresidue and/or at least one serine residue. In some embodiments thelinker sequence consists of glycine and serine residues. In someembodiments the linker sequence comprises or consists of alternatingglycine and serine residues (i.e. “GSGS” (SEQ ID NO:92) . . . etc.). Insome embodiments, the linker sequence has a length of 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15 or 1-20 amino acids.

Flexible linker sequences provided between plural sequences of a mutantp53 polypeptide within an antigen sequence according to the inventionallows flexible relative arrangement of the mutant p53 polypeptidesequences. In some embodiments, linker sequence(s) between mutant p53polypeptide sequences comprise or consist of glycine and serineresidues. In some embodiments, the linker sequences comprise or consistof alternating glycine and serine residues. In some embodiments, thelinker sequences comprise or consist of alternating glycine and serineresidues of 1-2, 1-3, 1-4 or 1-5 amino acids in length. In someembodiments, linker sequences between mutant p53 polypeptide sequencescomprise or consist of the sequence GSG (SEQ ID NO:89).

The immunogen may in addition to the antigen sequence, scaffold sequenceand any linker sequence(s) comprise further amino acids or amino acidsequences. For example, the immunogen may comprise amino acidsequence(s) to facilitate expression, folding, trafficking, processingor purification of the immunogen. For example, the immunogen maycomprise a sequence encoding a His, (e.g. 6×His), Myc GST, MBP, FLAG,HA, E, or Biotin tag, optionally at the N- or C-terminus.

Exemplary Embodiments of Immunogens

The present invention provides an immunogen comprising: (i) an antigensequence, comprising an amino acid sequence of the R175H mutant p53polypeptide including the R175H mutation and at least one amino acideither side of the mutation, and (ii) a scaffold sequence for providingthe antigen sequence in a solvent-accessible configuration. In someembodiments, the immunogen comprises more than sequence of the R175Hmutant p53 polypeptide; in some embodiments the immunogen comprises e.g.2, 3, 4 or 5 such sequences. In some embodiments, the immunogencomprises an antigen sequence comprising sequences of the R175H mutantp53 polypeptide (including the R175H mutation) of different lengths. Insome embodiments, the antigen sequence comprises sequence(s) of theR175H mutant p53 polypeptide of from 5 to 22, 7 to 20 or 9 to 18 aminoacids in length. In some embodiments, the immunogen comprises an antigensequence comprising one or more of: QHMTEVVRHCPHHERCSD (SEQ ID NO:77),TEVVRHCPHHER (SEQ ID NO:78) and VRHCPHHER (SEQ ID NO:79). In someembodiments the immunogen comprises an antigen sequence comprising SEQID NOs: 77, 78 and 79. In some embodiments, the immunogen comprises anantigen sequence comprising SEQ ID NOs: 77, 78 and 79, and comprisinglinker sequences as described hereinabove between SEQ ID NOs: 77, 78 and79. In some embodiments, the immunogen comprises an antigen sequencecomprising SEQ ID NOs: 77, 78 and 79 and linker sequences between thebetween the antigen sequence and scaffold sequence. In some embodimentsthe immunogen comprises, or consists of the following sequence:

(SEQ ID NO: 80) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGQHMTEVVRHCPHHERCSDGSGTEVVRHCPHHERGSGVRHCPHHERGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHH

The present invention provides an immunogen comprising: (i) an antigensequence, comprising an amino acid sequence of the R248Q mutant p53polypeptide including the R248Q mutation and at least one amino acideither side of the mutation, and (ii) a scaffold sequence for providingthe antigen sequence in a solvent-accessible configuration. In someembodiments, the immunogen comprises more than sequence of the R248Qmutant p53 polypeptide; in some embodiments the immunogen comprises e.g.2, 3, 4 or 5 such sequences. In some embodiments, the immunogencomprises an antigen sequence comprising sequences of the R248Q mutantp53 polypeptide (including the R248Q mutation) of different lengths. Insome embodiments, the antigen sequence comprises sequence(s) of theR248Q mutant p53 polypeptide of from 10 to 22, 12 to 20 or 15 to 18amino acids in length. In some embodiments, the immunogen comprises anantigen sequence comprising one or more of: SCMGGMNQRPILTIITLED (SEQ IDNO:81), MGGMNQRPILTIITLED (SEQ ID NO:82) and NSSCMGGMNQRPILT (SEQ IDNO:83). In some embodiments the immunogen comprises an antigen sequencecomprising SEQ ID NOs: 81, 82 and 83. In some embodiments, the immunogencomprises an antigen sequence comprising SEQ ID NOs: 81, 82 and 83, andcomprising linker sequences as described hereinabove between SEQ ID NOs:81, 82 and 83. In some embodiments, the immunogen comprises an antigensequence comprising SEQ ID NOs: 81, 82 and 83 and linker sequencesbetween the between the antigen sequence and scaffold sequence. In someembodiments the immunogen comprises, or consists of the followingsequence:

(SEQ ID NO: 84) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGSCMGGMNQRPILTIITLEDGSGMGGMNQRPILTIITLEDGSGNSSCMGGMNQRPILTGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHH

The present invention provides an immunogen comprising: (i) an antigensequence, comprising an amino acid sequence of the R273H mutant p53polypeptide including the R273H mutation and at least one amino acideither side of the mutation, and (ii) a scaffold sequence for providingthe antigen sequence in a solvent-accessible configuration. In someembodiments, the immunogen comprises more than sequence of the R273Hmutant p53 polypeptide; in some embodiments the immunogen comprises e.g.2, 3, 4 or 5 such sequences. In some embodiments, the immunogencomprises an antigen sequence comprising sequences of the R273H mutantp53 polypeptide (including the R273H mutation) of different lengths. Insome embodiments, the antigen sequence comprises sequence(s) of theR273H mutant p53 polypeptide of from 5 to 20, 6 to 18 or 10 to 15 aminoacids in length. In some embodiments, the immunogen comprises an antigensequence comprising one or more of: RNSFEVHVCA (SEQ ID NO:85),NLLGRNSFEVHVCAC (SEQ ID NO:86) and GRNSFEVHVCACP (SEQ ID NO:87). In someembodiments the immunogen comprises an antigen sequence comprising SEQID NOs: 85, 86 and 87. In some embodiments, the immunogen comprises anantigen sequence comprising SEQ ID NOs: 85, 86 and 87, and comprisinglinker sequences as described hereinabove between SEQ ID NOs: 85, 86 and87. In some embodiments, the immunogen comprises an antigen sequencecomprising SEQ ID NOs: 85, 86 and 87 and linker sequences between thebetween the antigen sequence and scaffold sequence. In some embodimentsthe immunogen comprises, or consists of the following sequence:

(SEQ ID NO: 88) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGRNSFEVHVCAGSGNLLGRNSFEVHVCACGSGGRNSFEVHVCACPGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHH

Methods for Producing an Antibody

The present invention provides methods for producing an antibody. Themethods typically involve introducing material into an animal which isrecognised by the immune system of the animal to be foreign (i.e.non-host), resulting in the selective production by the animal ofantibodies capable of binding to the material.

The material used to provoke an immune response is referred to herein asan immunogen. The immunogen may comprise or consist of, or may beprocessed to, an antigen. As used herein, “antigen” refers to a moleculecapable of stimulating the production of an antibody by stimulating anadaptive immune response, in particular the B lymphocyte mediated(humoral) adaptive immune response. The term “antigenic” as used hereinrefers to the ability of a molecule to stimulate the production ofantibodies by stimulating an adaptive immune response, in particular a Blymphocyte mediated adaptive immune response.

In the present invention, an immunogen may be a peptide or polypeptidemolecule.

The ability of animal immune system to produce antibodies capable ofbinding specifically to antigens can be used to generate antibodies fordetecting molecules of interest in various research, diagnostic andtherapeutic applications.

Methods for producing antibodies by immunization of animals are wellknown in the art, and are described, for example, in Antibodies: ALaboratory Manual, Second Edition, 2014; Edward A. Greenfield, ColdSpring Harbor Laboratory Press, which is hereby incorporated byreference in its entirety. In particular, Chapter 6 provides detaileddescription of the immunization of animals for the production ofantibodies.

Antibody production involves challenging an animal with an immunogen tostimulate production of antigen-specific antibodies which can then berecovered from the animal.

As used herein, “producing” an antibody refers to the process ofcreating an antibody, which may include one or more of, for example:preparing immunogen for immunization, immunizing an animal, hybridomaformation, collecting antibody, screening for binding to target,isotyping antibody, purifying antibody, or labelling antibody.

In the methods of the present invention antibodies are produced byimmunizing an animal with an immunogen. Any animal capable of producingantibody in response to immunization with immunogen is useful inaccordance with the present methods. It will be appreciated that themethods of the invention are not methods for the diagnosis or treatmentof diseases, nor methods of surgery. In some embodiments, the animal maybe a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or otherrodent (including any animal in the order Rodentia), cat, dog, pig,sheep, goat, cattle (including cows, e.g. dairy cows, or any animal inthe order Bos), horse (including any animal in the order Equidae),donkey, and non-human primate). In particular, the animal may be a mouse(e.g. Mus musculus), rat (e.g. Rattus norvegicus), or rhesus macaque(Macaca mulatta). In particular embodiments of the animal is a mouse. Inthe present methods, immunogen is administered to animals by way ofimmunization.

Immunization of an animal with an immunogen according to the presentinvention can be by any suitable means, such as those described, forexample, in Antibodies: A Laboratory Manual, Second Edition, 2014;Edward A. Greenfield, Cold Spring Harbor Laboratory Press (herebyincorporated by reference in entirety), in particular at Chapter 6.Materials for immunizations may be formulated as appropriate. Forexample, immunogen peptide/polypeptide may be diluted in sterile saline,and may combined with an adjuvant (e.g. Complete or Incomplete Freund'sAdjuvant and/or CpG) to form a stable emulsion. Appropriate amounts ofimmunogen for an individual immunization can be readily determined bythe skilled person, e.g. by reference to Antibodies: A LaboratoryManual, Second Edition, 2014; Edward A. Greenfield, Cold Spring HarborLaboratory Press (incorporated by reference herein above). Appropriatevolumes and concentrations for immunizations can also be determined bythe skilled person.

The method for producing an antibody according to the present inventionmay comprise a step of screening for the production of antibody; forexample, at an appropriate period of time following administration, asample (e.g. a blood sample) may be obtained from the animal andanalysed for antibody production. Immunoassays for the detection andquantification of antibody production are well known to the skilledperson, and are described, for example, in Antibodies: A LaboratoryManual, Second Edition, 2014; Edward A. Greenfield, Cold Spring HarborLaboratory Press, 2014 (incorporated by reference herein above), inparticular at Chapter 15. For example, a blood, plasma, serum or ascitessample may be collected from the animal, and analysed e.g. by ELISA orflow cytometry.

Antibodies produced by the methods according to the invention can beanalysed for binding to mutant p53 polypeptide and/or wildtype p53polypeptide by methods known to the skilled person. For example,analysis may be performed by ELISA, immunoblot (e.g. western blot), flowcytometry, immunohistochemistry, immunoprecipitation, surface plasmonresonance (BIAcore), biolayer interferometry.

Analysis may also include analysis of epitopes for antibodies producedby the methods, and may include analysis to determine the identity (e.g.the sequence) of antibodies. Such analysis may include antibodysequencing, e.g. by mass spectrometry.

In some embodiments the methods for producing an antibody may includeisolating antibodies capable of binding to the mutant p53 polypeptide.In some embodiments, the one or more antibodies are isolated from ananimal. The antibodies may be recovered from e.g. the blood, plasma,serum, or ascites of the animal. In some embodiments, antibodies may beisolated from cells obtained from an animal which has been immunizedwith immunogen in accordance with the invention. In some embodiments,the cells are B lymphocytes. In some embodiments, the antibodies may beisolated from cell culture supernatant from B lymphocytes cultured invitro.

Polyclonal antibodies may be recovered directly from blood or serumobtained from the animal at an appropriate time following administrationaccording to the invention.

In some embodiments, antibodies are obtained from a hybridoma producingone or more antibodies capable of binding to a mutant p53 polypeptide.In some embodiments, the antibodies are obtained from cell culturesupernatant of a culture of a hybridoma. In some embodiments, theantibodies are obtained from the blood, plasma, serum, or ascites of ananimal immunized with a hybridoma producing one or more antibodiescapable of binding to the mutant p53 polypeptide of interest.

Methods for isolating (i.e. purifying) antibodies from an antibodycontaining sample (e.g. cell, cell extract, cell culture medium, blood,plasma, serum, ascites) are well known to the skilled person, and aredescribed in detail in Antibodies: A Laboratory Manual, Second Edition,2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press(incorporated by reference herein above), in particular at Chapter 10.The methods include, for example, ion exchange chromatography, protein Aor protein G based purification, gel electrophoresis, dialysis, andaffinity purification based on target binding.

In some embodiments, the methods of the present invention compriseproducing a hybridoma producing one or more antibodies capable ofbinding to the mutant p53 polypeptide of interest. The methods comprisefusing a cell capable of producing one more antibodies capable ofbinding to the mutant p53 polypeptide of interest isolated from theanimal with a myeloma cell, to produce a hybridoma.

In particular, the methods of the present invention are for producingmonoclonal antibodies capable of binding to a mutant p53 polypeptide.This is achieved by immunizing an animal with immunogen according to theinvention, and subsequently generating monoclonal hybridomas from cellsisolated from the animals, the hybridomas producing antibodies of asingle type (i.e. of a single specificity), which are capable of bindingto the mutant p53 polypeptide.

Methods for hybridoma formation are well known to the skilled person,and are described, for example, in Antibodies: A Laboratory Manual,Second Edition, 2014; Edward A. Greenfield, Cold Spring HarborLaboratory Press (incorporated by reference herein above); in particularat Chapter 7. Briefly, an animal is immunized in accordance with theinvention, stimulating an adaptive immune response, and B lymphocytesare isolated from the animal and fused with a suitable myeloma cell lineto produce a hybridoma. B lymphocytes and myeloma cells may be fusedusing methods known to the skilled person. For example, cells may befused by co-centrifugation in polyethylene glycol (PEG). Followingfusion, cells are plated-out by limiting dilution in tissue cultureplates to roughly a single cell per well, and cultured in vitro.Hybridomas may be selected by culture using selective media known in theart (e.g. media containing hypoxanthine-aminopterin-thymidine (HAT)),which unfused lymphocytes and myeloma cells are unable to grow orsurvive in.

Antibodies produced by the methods of the invention may be produced on alarge scale using methods known to the skilled person. Hybridomas can bepropagated either in in vitro culture using standard methods of cellculture, or in vivo, e.g. as ascites in a host animal. In someembodiments, the methods of the present invention comprise propagatingthe hybridoma by in vitro cell culture. In some embodiments, the methodscomprise propagating the hybridoma in vivo by injecting a host animalwith the hybridoma.

Antibodies according to the invention can also be producedrecombinantly, as described hereinbelow. For example, a polynucleotideencoding a monoclonal antibody can be isolated from a B cell orhybridoma cell producing an antibody, e.g., by reverse transcription PCR(RT-PCR) using oligonucleotide primers that specifically amplify thegenes encoding the heavy and light chains of the antibody, and thesequence of the polynucleotide can be determined. Isolatedpolynucleotides encoding the heavy and light chains can be cloned intosuitable expression vectors which produce the monoclonal antibodies whentransfected into host cells such as E. coli, simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin proteins.

Antibodies

The antibodies of the present invention are specific for mutant p53polypeptides over wildtype p53 polypeptide.

The antibodies may be capable of binding to particular mutant p53polypeptides, and may not be capable of binding to wildtype p53polypeptide. By “not capable of binding”, we mean that the antibodiesexhibit substantially no binding to the peptide/polypeptide, or do notexhibit significant binding to the peptide/polypeptide, e.g. abovenon-specific binding levels.

As used herein, an antibody which is “specific for” a givenpolypeptide/peptide displays specific binding to thepolypeptide/peptide. “Specific binding” is interaction which is notnon-specific. Specific binding is mediated by non-covalent interactionssuch as Van der Waals forces, electrostatic interactions, hydrogenbonding, and hydrophobic interactions. An antibody of the presentinvention preferably displays specific binding to the mutant p53polypeptide for which the antibody is specific over wildtype p53 and/orother, different, mutants of p53.

An antibody which is specific for a given peptide/polypeptide overanother, reference peptide/polypeptide may bind to the givenpeptide/polypeptide with greater affinity than the affinity of bindingof the antibody to the reference peptide/polypeptide. An antibodydisplaying ‘greater affinity’ for a given peptide/polypeptide binds tothat peptide/polypeptide with greater strength as compared to thestrength of binding to the reference peptide/polypeptide. An antibody ofthe present invention may bind to the mutant p53 polypeptide for whichthe antibody is specific with greater affinity as compared to theaffinity of binding by the antibody to wildtype p53 and/or other,different, mutants of p53.

The affinity of an antibody for its antigen refers to the strength withwhich an antibody binds to the antigen. Antibody affinity can bedetermined by methods well known to the person skilled in the art. Suchmethods include, for example, in vitro analysis by SPR assay withpurified antigen. Binding can be expressed in the following terms: “onrate” k_(on), a constant representing how quickly the antibody/antigencomplex forms; “off rate” k_(off), a constant representing how quicklythe antibody/antigen complex dissociates; and the equilibriumdissociation constant K_(D), calculated according to the formulaK_(D)=(K_(off)/K_(on)).

The affinity of binding of an antibody for a peptide/polypeptide can bedetermined quantitatively. In some embodiments, an antibody displayinggreater affinity for a given peptide/polypeptide as compared to areference peptide/polypeptide binds to the given peptide/polypeptidewith a K_(D) value or an EC50 value which is less than the value forbinding of the antibody to the reference peptide/polypeptide.

“Cross-reactivity” refers to the ability of an antibody to bind to morethan one peptide/polypeptide. That is, an antibody displaying binding totwo or more peptide/polypeptides is said to be “cross-reactive” forthose peptides/polypeptides. An antibody according to the presentinvention preferably displays low or no cross-reactivity to wildtype p53and/or mutants of p53 other than the mutant p53 polypeptide for whichthe antibody is specific. By “not cross-reactive” we mean that anantibody exhibits low cross-reactivity, no cross-reactivity orsubstantially no binding to the respective peptide/polypeptide.

The antibodies according to the present invention display greateraffinity for a given mutant p53 polypeptide as compared to affinity forwildtype p53 polypeptide. In some embodiments, the antibodies displaygreater affinity for a given mutant of human p53 polypeptide as comparedto affinity for wildtype human p53 polypeptide. In some embodiments theantibodies display greater affinity for a p53 peptide/polypeptidecomprising a mutation corresponding to one of R175H, R248Q, R273H,R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y, H179R, Y220C orR337H as compared to affinity for a p53 peptide/polypeptide notcomprising said mutation. In some embodiments, an antibody may displaygreater affinity for a peptide/polypeptide comprising the amino acidsequence of one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 or a fragment thereof comprising the mutation, as compared toaffinity for a peptide/polypeptide comprising the amino acid sequence ofSEQ ID NO:1, or a fragment thereof.

The antibodies according to the present invention may display greateraffinity for a given mutant p53 polypeptide as compared to other mutantp53 polypeptides. In some embodiments, the antibodies display greateraffinity for a given mutant of human p53 polypeptide as compared toaffinity for other mutants of human p53 polypeptide.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R175H ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:3, or a fragment thereof comprising the R175H mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R248Q ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:4, or a fragment thereof comprising the R248Q mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R273H ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R248W, G245S, R273C, R282W, R249S G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:5, or a fragment thereof comprising the R273H mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R248W ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, G245S, R273C, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:6, or a fragment thereof comprising the R248W mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to G245S ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, R273C, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:7, or a fragment thereof comprising the G245S mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R273C ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:8, or a fragment thereof comprising the R273C mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R282W ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R249S, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:9, or a fragment thereof comprising the R282W mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R249S ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, G245D, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:10, or a fragment thereof comprising the R249S mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to G245D ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, C176F, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:11, or a fragment thereof comprising the G245D mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to C176F ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, H179Y,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:12, or a fragment thereof comprising the C176F mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to H179Y ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F,H179R, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:13, or a fragment thereof comprising the H179Y mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to H179R ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F,H179Y, Y220C or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:14, or a fragment thereof comprising the H179R mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 15 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to Y220C ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F,H179Y, H179R or R337H.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:15, or a fragment thereof comprising the Y220C mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 16.

In some embodiments the antibodies display greater affinity for a p53peptide/polypeptide comprising a mutation corresponding to R337H ascompared to affinity for a p53 peptide/polypeptide not comprising saidmutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F,H179Y, H179R or Y220C.

In some embodiments, an antibody may display greater affinity for apolypeptide/peptide comprising or consisting of the amino acid sequenceof SEQ ID NO:16, or a fragment thereof comprising the R337H mutation, ascompared to a polypeptide/peptide comprising or consisting of the aminoacid sequence of one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15.

The present invention is concerned in particular with antibodies whichare capable of binding to mutant p53 polypeptides, which are not capableof binding to wildtype p53 polypeptide. That is, the invention isconcerned with antibodies which bind specifically to mutant p53polypeptides, and which are not cross-reactive with (i.e. do not alsobind to) wildtype p53 polypeptide.

The present invention is also concerned with antibodies which arecapable of binding to a given, particular p53 mutant and which are notcapable of binding to other, different mutants of p53. That is, theinvention is concerned with antibodies which bind specifically to agiven, particular p53 mutant, and which are not cross-reactive with(i.e. do not also bind to) other, different mutants of p53.

In some embodiments, an antibody may be capable of binding to a givenmutant p53 polypeptide, and may not be capable of binding to anothermutant p53 polypeptide.

Whether an antibody is capable of binding to a given molecule can bedetermined by methods well known to the skilled person. For example, themethods include analysis by immunoassay such as enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, western blot,immunofluorescence methods etc., and other methods such as surfaceplasmon resonance (SPR) assay and fluorescence resonance energy transfer(FRET).

The ability of an antibody to bind to a given peptide or polypeptide canbe determined by detection of interaction between the antibody andpeptide/polypeptide which is of greater affinity than non-specificinteraction between the antibody and a control peptide/polypeptide. Acontrol peptide/polypeptide may be a peptide or polypeptide to which itis known, or to which is has been determined, the antibody does notspecifically bind.

In some embodiments, the capability of an antibody to bind to a givenpeptide or protein can be determined by observation of interactionbetween the antibody and peptide/polypeptide which is stronger than,more stable than, lasts longer than, or has a lower equilibriumdissociation constant (K_(D)) than non-specific interaction between theantibody and a negative control peptide/polypeptide.

In some embodiments, the extent of binding of an antibody to anunrelated target is less than about 10% of the binding of the antibodyto the target as measured, e.g., by ELISA, SPR, Bio-Layer Interferometryor by RIA. Alternatively, the binding specificity may be reflected interms of binding affinity where an antibody according to the presentinvention binds to its cognate mutant p53 polypeptide with a K_(D) thatis at least 0.1 order of magnitude (i.e. 0.1×10^(n), where n is aninteger representing the order of magnitude) greater than the K_(D) ofthe antibody towards another target molecule (e.g. other mutant p53polypeptide and/or wildtype p53 polypeptide). This may optionally be oneof at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

In some embodiments of the present invention, the methods produce anantibody capable of binding to an antigen sequence in the context of thefull-length amino acid sequence of a mutant p53 polypeptide. That is, insome embodiments, the methods produce an antibody which can bind to themutant p53 polypeptide through binding to the antigen sequence.

The site within a peptide/polypeptide to which an antibody binds can bedetermined by the skilled person using various methods well known in theart, including X-ray co-crystallography analysis of antibody-antigencomplexes, peptide scanning, mutagenesis mapping, hydrogen-deuteriumexchange analysis by mass spectrometry, phage display, competition ELISAand proteolysis-based ‘protection’ methods. Such methods are described,for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which ishereby incorporated by reference in its entirety.

Preferably, the antibodies bind to the mutant p53 polypeptide for whichthey are specific with a KD of 1 μM or less, preferably one of ≤100 nM,≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM,≤8 nM, nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤500 pM.Binding affinity of an antibody for its target is often described interms of its dissociation constant (K_(D)). Binding affinity can bemeasured by methods known in the art, such as by ELISA, Surface PlasmonResonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012)907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) JBiomol Screen 20(4): 498-507), or by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of the antibody and antigenmolecule.

Antibodies according to the present invention may be provided inisolated and/or purified form.

By “antibody” we include a fragment or derivative thereof, or asynthetic antibody or synthetic antibody fragment.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). Chimeric antibodies arediscussed by Neuberger et al (1988, 8th International BiotechnologySymposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are useful in the methods of the inventionand are a homogenous population of antibodies specifically targeting asingle epitope on an antigen.

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen binding fragments of antibodies, such as Fab and Fab₂ fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (V_(H)) and variable light(V_(L)) domains of the antibody/fragment are involved in antigenrecognition, a fact first recognised by early protease digestionexperiments. Further confirmation was found by “humanisation” of rodentantibodies. Variable domains of rodent origin may be fused to constantdomains of human origin such that the resultant antibody retains theantigenic specificity of the rodent parent antibody (Morrison et al(1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodiesmay also be made using phage display technology as is well known in theart.

The present application also provides antibodies, or antigen bindingfragments, optionally isolated, which are capable of binding to a givenmutant p53 polypeptide, which are bispecific antibodies/antigen bindingfragments comprising (i) an antigen binding fragment or polypeptideaccording to the invention, and (ii) an antigen binding fragment capableof binding to a polypeptide other than the given mutant p53 polypeptide.

In some embodiments, the antigen binding fragment capable of binding toa polypeptide other than the given mutant p53 polypeptide may be capableof binding to another, different mutant p53 polypeptide.

An antigen binding fragment of a bispecific antibody or bispecificantigen binding fragment according to the present invention may be anyfragment of a polypeptide which is capable of binding to an antigen. Insome embodiments, an antigen binding fragment comprises at least thethree light chain CDRs (i.e. LC-CDR1, LC-CDR2 and LC-CDR3) and threeheavy chain CDRs (i.e. HC-CDR1, HC-CDR2 and HC-CDR3) which togetherdefine the antigen binding region of an antibody or antigen bindingfragment. In some embodiments, an antigen binding fragment may comprisethe light chain variable domain and heavy chain variable domain of anantibody or antigen binding fragment. In some embodiments, an antigenbinding fragment may comprise the light chain polypeptide and heavychain polypeptide of an antibody or antigen binding fragment.

Bispecific antibodies and bispecific antigen binding fragments accordingto the invention may be provided in any suitable format, such as thoseformats described in Kontermann MAbs 2012, 4(2): 182-197, which ishereby incorporated by reference in its entirety. For example, abispecific antibody or bispecific antigen binding fragment may be abispecific antibody conjugate (e.g. an IgG2, F(ab)₂ or CovX-Body), abispecific IgG or IgG-like molecule (e.g. an IgG, scFv₄-Ig, IgG-scFv,scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb², or Tandemab commonLC), an asymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG,kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair orSEED-body), a small bispecific antibody molecule (e.g. a Diabody (Db),dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triplebody, triple head, Fab-scFv, or F(ab′)₂-scFv₂), a bispecific Fc andC_(H)3 fusion protein (e.g. a taFv-Fc, Di-diabody, scDb-C_(H)3,scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-C_(H)3), or abispecific fusion protein (e.g. a scFv₂-albumin, scDb-albumin,taFv-toxin, DNL-Fab₃, DNL-Fab₄-IgG, DNL-Fab₄-IgG-cytokine₂). See inparticular FIG. 2 of Kontermann MAbs 2012, 4(2): 182-19.

The skilled person is able to design and prepare bispecific antibodiesand bispecific antigen binding fragments according to the presentinvention. Methods for producing bispecific antibodies includechemically crosslinking of antibodies or antibody fragments, e.g. withreducible disulphide or non-reducible thioether bonds, for example asdescribed in Segal and Bast, 2001. Production of Bispecific Antibodies.Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which ishereby incorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH-groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers. Other methodsfor producing bispecific antibodies include fusing antibody-producinghybridomas e.g. with polyethylene glycol, to produce a quadroma cellcapable of secreting bispecific antibody, for example as described in D.M. and Bast, B. J. 2001. Production of Bispecific Antibodies. CurrentProtocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments accordingto the present invention can also be produced recombinantly, byexpression from e.g. a nucleic acid construct encoding polypeptides forthe antigen binding molecules, for example as described in AntibodyEngineering: Methods and Protocols, Second Edition (Humana Press, 2012),at Chapter 40: Production of Bispecific Antibodies: Diabodies and TandemscFv (Hornig and Farber-Schwarz), or French, How to make bispecificantibodies, Methods Mol. Med. 2000; 40:333-339, the entire contents ofboth of which are hereby incorporated by reference. For example, a DNAconstruct encoding the light and heavy chain variable domains for thetwo antigen binding fragments (i.e. the light and heavy chain variabledomains for the antigen binding fragment capable of binding to a mutantp53 polypeptide, and the light and heavy chain variable domains for theantigen binding fragment capable of binding to another target protein),and including sequences encoding a suitable linker or dimerizationdomain between the antigen binding fragments can be prepared bymolecular cloning techniques. Recombinant bispecific antibody canthereafter be produced by expression (e.g. in vitro) of the construct ina suitable host cell (e.g. a mammalian host cell), and expressedrecombinant bispecific antibody can then optionally be purified.

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al,J. Mol. Biol. 226:889-896 (1992).

Antibodies according to the present invention preferably exhibitspecific binding to a given mutant p53 polypeptide. An antibody thatspecifically binds to a target molecule preferably binds the target withgreater affinity, and/or with greater duration than it binds to othertargets. The present antibodies may bind with greater affinity to agiven mutant p53 polypeptide than to other mutant p53 polypeptides andwildtype p53 polypeptide.

The antibodies, fragments or polypeptides may display substantially nobinding to wildtype p53 polypeptide, e.g. human p53 polypeptide. This isan unexpected feature for antibodies directed against mutant p53polypeptides as described herein, which may differ from wildtype p53polypeptide amino acid sequence by only a single amino acid residue.That is, it would be expected that an antibody capable of binding to apolypeptide would display cross-reactivity for another polypeptidediffering by only a single amino acid. The present antibodies which arespecific for single acid mutants of p53 are useful to discriminatemutant from wildtype p53, and therefore have wide range of uses, such asin research, therapy and diagnostic applications.

‘Substantially no binding’ as used herein refers to binding which is notsignificantly greater than the level of binding by a negative controlantibody (e.g. an antibody directed against a target unrelated towildtype p53 polypeptide, or an antibody known not to bind to wildtypep53 polypeptide). In some embodiments, an antibody according to thepresent invention may exhibit binding to wildtype p53 polypeptide (e.g.human wildtype p53 polypeptide) which is ≤500%, ≤300%, ≤250%, ≤200%,≤150%, or ≤100% of the binding to wildtype p53 polypeptide displayed bya negative control antibody (e.g. an antibody directed against a targetunrelated to wildtype p53 polypeptide, or an antibody known not to bindto wildtype p53 polypeptide), in a given assay or at a givenconcentration. Binding can be measured by techniques well known to theperson skilled in the art, including ELISA, SPR, Bio-LayerInterferometry, flow cytometry or by a radioimmunoassay (RIA).

In some embodiments, an antibody according to the invention may becapable of inhibiting tumour growth or cancer progression. In someembodiments an antibody according to the invention may displayanti-cancer activity. In some embodiments, inhibition of tumour growthor cancer progression may be in vivo. ‘Inhibition’ may be reduction orcontrol of tumour growth, or reduction or control of the number ofcancer cells. Inhibition of tumour growth or cancer progression can beevaluated in vivo, for example in an animal model of a cancer asdescribed herein.

Anti-cancer activity may be determined by detection of a reduced numberof tumor cells and/or reduced tumor volume following treatment with anantibody/fragment according to the invention, as compared to the numberof tumor cells and/or tumor volume in the absence of treatment, ortreatment with a negative control antibody.

In some embodiments, the antibody according to the invention is capableof binding to R175H p53 polypeptide, e.g. as shown in SEQ ID NO:3, or afragment thereof comprising the R175H mutation. In some embodiments, theantibody according to the invention is capable of binding to R248Q p53polypeptide, e.g. as shown in SEQ ID NO:4, or a fragment thereofcomprising the R248Q mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R273H p53polypeptide, e.g. as shown in SEQ ID NO:5, or a fragment thereofcomprising the R273H mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R248W p53polypeptide, e.g. as shown in SEQ ID NO:6, or a fragment thereofcomprising the R248W mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to G245S p53polypeptide, e.g. as shown in SEQ ID NO:7, or a fragment thereofcomprising the G245S mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R273C p53polypeptide, e.g. as shown in SEQ ID NO:8, or a fragment thereofcomprising the R273C mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R282W p53polypeptide, e.g. as shown in SEQ ID NO:9, or a fragment thereofcomprising the R282W mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R249S p53polypeptide, e.g. as shown in SEQ ID NO:10, or a fragment thereofcomprising the R249S mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to G245D p53polypeptide, e.g. as shown in SEQ ID NO:11, or a fragment thereofcomprising the G245D mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to C176F p53polypeptide, e.g. as shown in SEQ ID NO:12, or a fragment thereofcomprising the C176F mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to H179Y p53polypeptide, e.g. as shown in SEQ ID NO:13, or a fragment thereofcomprising the H179Y mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to H179R p53polypeptide, e.g. as shown in SEQ ID NO:14, or a fragment thereofcomprising the H179R mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to Y220C p53polypeptide, e.g. as shown in SEQ ID NO:15, or a fragment thereofcomprising the Y220C mutation. In some embodiments, the antibodyaccording to the invention is capable of binding to R337H p53polypeptide, e.g. as shown in SEQ ID NO:16, or a fragment thereofcomprising the R337H mutation.

In some embodiments, an antibody according to the invention is specificfor a particular mutant p53 polypeptide. That is, in some embodiments anantibody is capable of binding only to a particular mutant p53polypeptide and is not capable of binding to a p53 polypeptide otherthan said mutant p53 polypeptide such as wildtype p53 polypeptide orother mutant p53 polypeptide.

In some embodiments, the antibodies of the present invention are capableof binding to a given mutant of human p53 polypeptide and are notcapable of binding to wildtype human p53 polypeptide. In someembodiments the antibodies are capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to one of R175H,R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F, H179Y,H179R, Y220C, or R337H, and are not capable of binding to a p53peptide/polypeptide not comprising said mutation. In some embodiments,an antibody may be capable of binding to a peptide/polypeptidecomprising the amino acid sequence of one of SEQ ID NOs: 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or 16, or a fragment thereof comprising themutation, and not capable of binding to peptide/polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, or a fragment thereof.

The antibodies according to the present invention may be capable ofbinding to a given mutant p53 polypeptide, and may not be capable ofbinding to other mutant p53 polypeptides. In some embodiments, theantibodies may be capable of binding to a given mutant of human p53polypeptide and may not be capable of binding to other mutants of humanp53 polypeptide.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R175H, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:3, or a fragmentthereof comprising the mutation, and may not be capable of binding to toa polypeptide/peptide comprising or consisting of the amino acidsequence of one of SEQ ID NO:1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R248Q, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R273H, R248W, G245S, R273C, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:4, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R273H, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R248W, G245S, R273C, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:5, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R248W, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, G245S, R273C, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:6, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to G245S, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, R273C, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:7, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R273C, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R282W, R249S, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:8, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R282W, andmay not be capable of binding a p53 peptide/polypeptide not comprisingsaid mutation, or comprising a mutation corresponding to one or more ofR175H, R248Q, R273H, R248W, G245S, R273C, R249S, G245D, C176F, H179Y,H179R, Y220C, or R337H. In some embodiments, an antibody may be capableof binding to a polypeptide/peptide comprising or consisting of theamino acid sequence of SEQ ID NO:9, or a fragment thereof comprising themutation, and may not be capable of binding to a polypeptide/peptidecomprising or consisting of the amino acid sequence of one of SEQ IDNO:1, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to G245S, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, G245D,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:10, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to G245D, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,C176F, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:11, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to C176F, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,G245D, H179Y, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:12, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to H179Y, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,G245D, C176F, H179R, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:13, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to H179R, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,G245D, C176F, H179Y, Y220C, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:14, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to Y220C, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,G245D, C176F, H179Y, H179R, or R337H. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:15, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 16.

In some embodiments the antibodies may be capable of binding to a p53peptide/polypeptide comprising a mutation corresponding to R337H, andmay not be capable of binding to a p53 peptide/polypeptide notcomprising said mutation, or comprising a mutation corresponding to oneor more of R175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S,G245D, C176F, H179Y, H179R, or Y220C. In some embodiments, an antibodymay be capable of binding to a polypeptide/peptide comprising orconsisting of the amino acid sequence of SEQ ID NO:16, or a fragmentthereof comprising the mutation, and may not be capable of binding to apolypeptide/peptide comprising or consisting of the amino acid sequenceof one of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

A fragment of a mutant p53 polypeptide of the invention to which anantibody according to the invention is capable of binding comprises themutation. In some embodiments the fragment of a mutant p53 polypeptideof the invention to which an antibody according to the invention iscapable of binding may comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or 50 amino acids. Insome embodiments the fragment may consist of 5 to 300, 5 to 250, 5 to200, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 40, 5 to 30, 5 to 20, or5 to 10 amino acids of the amino acid sequence of the mutant p53polypeptide.

In some embodiments, an antibody according to the invention may bedefined by reference to the epitope of the mutant p53 polypeptide towhich the antibody binds. In some embodiments, the antibody may bind toa linear epitope, consisting of a contiguous sequence of amino acids(i.e. an amino acid primary sequence). In some embodiments, the antibodymay bind to a conformational epitope, consisting of a discontinuoussequence of amino acids of the amino acid sequence. The amino acids ofthe discontinuous sequence of amino acids may be located in differentregions of the mutant p53 polypeptide, and be positioned in closeproximity when the sequence is folded, e.g. into its native structure.

In some embodiments, the antibody capable of binding to R175H p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positions100 to 250, 110 to 240, 120 to 230, 130 to 220, 140 to 210, 150 to 200,160 to 190, or 170 to 180 of SEQ ID NO:3.

In some embodiments, an antibody capable of binding to R248Q p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 173 to 323, 183 to 313, 193 to 303, 203 to 293, 213 to 283, 223 to273, 233 to 263, or 243 to 253 of SEQ ID NO:4.

In some embodiments, an antibody capable of binding to R273H p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 198 to 348, 208 to 338, 218 to 328, 228 to 318, 238 to 308, 248 to298, 258 to 288, or 268 to 278 of SEQ ID NO:5.

In some embodiments, an antibody capable of binding to R248W p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 173 to 323, 183 to 313, 193 to 303, 203 to 293, 213 to 283, 223 to273, 233 to 263, or 243 to 253 of SEQ ID NO:6.

In some embodiments, an antibody capable of binding to G245S p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 170 to 320, 180 to 310, 190 to 300, 200 to 290, 210 to 280, 220 to270, 230 to 260, or 240 to 250 of SEQ ID NO:7.

In some embodiments, an antibody capable of binding to R273C p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 198 to 348, 208 to 338, 218 to 328, 228 to 318, 238 to 308, 248 to298, 258 to 288, or 268 to 278 of SEQ ID NO:8.

In some embodiments, an antibody capable of binding to R282W p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 207 to 357, 217 to 347, 227 to 337, 237 to 327, 247 to 317, 257 to301, 267 to 297 or 277 to 287 of SEQ ID NO:9.

In some embodiments, an antibody capable of binding to R249S p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 174 to 324, 184 to 314, 194 to 304, 204 to 294, 214 to 284, 224 to274, 234 to 264, or 244 to 254 of SEQ ID NO:10.

In some embodiments, an antibody capable of binding to G245D p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 170 to 320, 180 to 310, 190 to 300, 200 to 290, 210 to 280, 220 to270, 230 to 260, or 240 to 250 of SEQ ID NO:11.

In some embodiments, an antibody capable of binding to C176F p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 101 to 251, 111 to 241, 121 to 231, 131 to 221, 141 to 211, 151 to201, 161 to 191, or 171 to 181 of SEQ ID NO:12.

In some embodiments, an antibody capable of binding to H179Y p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 104 to 254, 114 to 244, 124 to 234, 134 to 224, 144 to 214, 154 to204, 164 to 194, or 174 to 184 of SEQ ID NO:13.

In some embodiments, an antibody capable of binding to H179R p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 104 to 254, 114 to 244, 124 to 234, 134 to 224, 144 to 214, 154 to204, 164 to 194, or 174 to 184 of SEQ ID NO:14.

In some embodiments, an antibody capable of binding to Y220C p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 145 to 295, 155 to 285, 165 to 275, 175 to 265, 185 to 255, 195 to245, 205 to 235, or 215 to 225 of SEQ ID NO:15.

In some embodiments, an antibody capable of binding to R337H p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, an amino acid sequence corresponding to one of positionsto 262 to 412, 272 to 402, 282 to 392, 292 to 382, 302 to 372, 312 to362, 322 to 352, or 332 to 342 of SEQ ID NO:16.

In some embodiments, the antibody capable of binding to R175H p53polypeptide may bind to an epitope of the polypeptide comprising, orconsisting of, amino acid positions corresponding to positions 175 to179 of SEQ ID NO:3, i.e. HCPHH (SEQ ID NO:68), or amino acid positionscorresponding to positions 175 (i.e. H) and 177 to 179 (i.e. PHH (SEQ IDNO:69)) of SEQ ID NO:3, or amino acid positions corresponding topositions 175 (i.e. H) and 178 to 179 (i.e. HH (SEQ ID NO:70)) of SEQ IDNO:3. That is, in some embodiments, the antibody capable of binding toR175H p53 polypeptide may bind to an epitope as follows:

Epitope Corresponding positions of SEQ ID NO: 3 HCPHH 175 to 179 H . . .PHH 175 and 177 to 179 H . . . HH 175 and 178 to 179

In some embodiments, an antibody capable of binding to R248Q p53polypeptide may bind to epitope of the polypeptide comprising, orconsisting of, amino acid positions corresponding to positions 247 to249 of SEQ ID NO:4, i.e. NQR (SEQ ID NO:71), or amino acid positionscorresponding to positions 215 to 216 (i.e. SV (SEQ ID NO:72)) and 233to 234 (i.e. HY (SEQ ID NO:73)) of SEQ ID NO:4, or amino acid positionscorresponding to positions 249 to 250 of SEQ ID NO:4, i.e. RP (SEQ IDNO:74). That is, in some embodiments, the antibody capable of binding toR248Q p53 polypeptide may bind to an epitope as follows:

Epitope Corresponding positions of SEQ ID NO: 4 NQR 247 to 249 SV . . .HY 215 to 216 and 233 to 234 RP 249 to 250

In some embodiments, an antibody capable of binding to R273H p53polypeptide may bind to epitope of the polypeptide comprising, orconsisting of, amino acid positions corresponding to positions 272 to273 of SEQ ID NO:5, i.e. VH (SEQ ID NO:75).

In some aspects, the antibody is clone anti-R175H p53 antibody clone 4H5or MH 4H5. Anti-R175H p53 antibody clones 4H5 and MH 4H5 comprise thefollowing CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 19) QSLLNSGNQKSY LC-CDR2:(SEQ ID NO: 20) GAS LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT Heavy chain:HC-CDR1: (SEQ ID NO: 25) GFTFTEYT HC-CDR2: (SEQ ID NO: 26) IDPNNGVTHC-CDR3: (SEQ ID NO: 27) ARWGGDYV

For all of the antibodies described herein, the CDRs are definedaccording to VBASE2 CDR prediction tool (Retter et al. Nucleic AcidsResearch (2005) 33 (Database issue):D671-674, hereby incorporated byreference in its entirety).

In some aspects, the antibody is clone anti-R175H p53 antibody clone 7B9or MH 7B9. Anti-R175H p53 antibody clones 7B9 and MH 7B9 comprise thefollowing CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 23) QSLLNSGNQKSN LC-CDR2:(SEQ ID NO: 20) GAS LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT Heavy chain:HC-CDR1: (SEQ ID NO: 29) GYTFTEYT HC-CDR2: (SEQ ID NO: 30) INPYSGGTHC-CDR3: (SEQ ID NO: 27) ARWGGDYV

In some aspects, the antibody is clone anti-R175H p53 antibody clone10C8 or MH 1008. Anti-R175H p53 antibody clones 1008 and MH 1008comprise the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 23) QSLLNSGNQKSN LC-CDR2:(SEQ ID NO: 20) GAS LC-CDR3: (SEQ ID NO: 21) QNDHSYPLT Heavy chain:HC-CDR1: (SEQ ID NO: 29) GYTFTEYT HC-CDR2: (SEQ ID NO: 30) INPYSGGTHC-CDR3: (SEQ ID NO: 27) ARWGGDYV

In some embodiments in accordance with the various aspects of thepresent invention, wherein HC-CDR2 is INPYSGGT (SEQ ID NO: 30), thissequence may be comprised in the sequence INPYSGGTV (SEQ ID NO: 76). Insome embodiments, HC-CDR2 is INPYSGGTV (SEQ ID NO: 76).

Anti-R175H p53 antibodies according to the present invention maycomprise the CDRs of clone 4H5, 7B9 or 1008 or one of SEQ ID NOs 18 or22; and 24 or 28.

Anti-R175H p53 antibodies according to the present invention maycomprise the CDRs of clone MH 4H5, MH 7B9 or MH 1008 or one of SEQ IDNOs 237 or 238; and 239, 240 or 241.

Amino acid sequences of the Wand V_(H) chains of anti-R175H p53 antibodyclones are shown in FIGS. 24, 25, 49 and 50 . The encoding nucleotidesequences are shown in FIGS. 27, 51 and 52 .

Anti-R175H p53 antibodies may have V_(L) and/or V_(H) chains comprisingan amino acid sequence that has a high percentage sequence identity toone or more of the V_(L) and/or V_(H) amino acid sequences of SEQ ID NOs18, 22, 237 and 238; or 24, 28, 239, 240 or 241, or to one of the aminoacid sequences shown in FIGS. 24, 25, 49 and 50 . For example,antibodies according to the present invention include antibodies thatbind to R175H p53 polypeptide and have a V_(L) or V_(H) chain thatcomprises an amino acid sequence having at least 70%, more preferablyone of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the V_(L) orV_(H) chain amino acid sequence of one of SEQ ID NOs 18, 22, 237, 238,24, 28, 239, 240 or 241, or to one or the amino acid sequences shown inFIGS. 24, 25, 49 and 50 .

In some aspects, the antibody is clone anti-R248Q p53 antibody clone3G11 or MH 3G11. Anti-R248Q p53 antibody clones 3G11 and MH 3G11comprise the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 41) QSLLYSDGKTY LC-CDR2:(SEQ ID NO: 42) LVS LC-CDR3: (SEQ ID NO: 43) WQGTHFPLT Heavy chain:HC-CDR1: (SEQ ID NO: 46) GYTFTDYY HC-CDR2: (SEQ ID NO: 47) IHPKNGGTHC-CDR3: (SEQ ID NO: 48) AKMGGYDDY

In some aspects, the antibody is clone anti-R248Q p53 antibody clone 4H2or MH 4H2. Anti-R248Q p53 antibody clones 4H2 and MH 4H2 comprise thefollowing CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 41) QSLLYSDGKTY LC-CDR2:(SEQ ID NO: 42) LVS LC-CDR3: (SEQ ID NO: 43) WQGTHFPLT Heavy chain:HC-CDR1: (SEQ ID NO: 46) GYTFTDYY HC-CDR2: (SEQ ID NO: 50) IDPKNGGTHC-CDR3: (SEQ ID NO: 51) AKQGGFDDY

Anti-R248Q p53 antibodies according to the present invention maycomprise the CDRs of clone 3G11 or 4H2, or one of SEQ ID NOs 40 or 44;and 45 or 49.

Anti-R248Q p53 antibodies according to the present invention maycomprise the CDRs of clone MH 3G11 or MH 4H2, or one of SEQ ID NOs 251or 252; and 253 or 254.

Amino acid sequences of the Wand V_(H) chains of anti-R248Q p53 antibodyclones are shown in FIGS. 28, 29, 57 and 58 . The encoding nucleotidesequences are shown in FIGS. 31, 59 and 60 .

Anti-R248Q p53 antibodies may have V_(L) and/or V_(H) chains comprisingan amino acid sequence that has a high percentage sequence identity toone or more of the V_(L) and/or V_(H) amino acid sequences of SEQ ID NOs40, 44, 251 and 252; or 45, 49, 253 or 254 or to one of the amino acidsequences shown in FIGS. 28, 29, 57 and 58 . For example, antibodiesaccording to the present invention include antibodies that bind to R248Qp53 polypeptide and have a V_(L) or V_(H) chain that comprises an aminoacid sequence having at least 70%, more preferably one of at least 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, sequence identity to the V_(L) or V_(H) chain aminoacid sequence of one of SEQ ID NOs 40, 44, 251, 252, 45, 49, 253 or 254or to one of the amino acid sequences shown in FIGS. 28, 29, 57 and 58 .

In some aspects, the antibody is clone anti-R273H p53 antibody clone13E4 or MH 13E4. Anti-R273H p53 antibody clones 13E4 and MH 13E4comprise the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 59) QSIVHNNGDTY LC-CDR2:(SEQ ID NO: 60) KVS LC-CDR3: (SEQ ID NO: 61) FQGSHLPLT Heavy chain:HC-CDR1: (SEQ ID NO: 63) GFSFSDYY HC-CDR2: (SEQ ID NO: 64) ISVGGTYTHC-CDR3: (SEQ ID NO: 65) VRDGNDGKFLG

Anti-R273H p53 antibodies according to the present invention maycomprise the CDRs of clone 13E4, or one of SEQ ID NOs 58 and 62.

Anti-R273H p53 antibodies according to the present invention maycomprise the CDRs of clone MH 13E4, or one of SEQ ID NOs 247 and 248.

Amino acid sequences of the Wand V_(H) chains of anti-R273H p53 antibodyclones are shown in FIGS. 32, 33, 53 and 54 . The encoding nucleotidesequences are shown in FIGS. 34, 55 and 56 .

Anti-R273H p53 antibodies may have V_(L) and/or V_(H) chains comprisingan amino acid sequence that has a high percentage sequence identity toone or more of the V_(L) and/or V_(H) amino acid sequences of SEQ ID NOs58, 247, 62 and 248, or to one or the amino acid sequences shown inFIGS. 32, 33, 53 and 54 . For example, antibodies according to thepresent invention include antibodies that bind to R273H p53 polypeptideand have a V_(L) or V_(H) chain that comprises an amino acid sequencehaving at least 70%, more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the V_(L) or V_(H) chain amino acid sequenceof one of SEQ ID NOs 58, 247, 62 and 248, or to one or the amino acidsequences shown in FIGS. 32, 33, 53 and 54 .

In an antibody according to the present invention one or two or three orfour of the six CDR sequences may vary. A variant may have one or twoamino acid substitutions in one or two of the six CDR sequences.

The light and heavy chain CDRs may also be particularly useful inconjunction with a number of different framework regions. Accordingly,light and/or heavy chains having LC-CDR1-3 or HC-CDR1-3 may possess analternative framework region. Suitable framework regions are well knownin the art and are described for example in M. Lefranc & G. Lefranc(2001) “The Immunoglobulin FactsBook”, Academic Press, incorporatedherein by reference.

Antibodies according to the present invention may be detectably labelledor, at least, capable of detection. For example, the antibody may belabelled with a radioactive atom or a coloured molecule or a fluorescentmolecule or a molecule which can be readily detected in any other way.Suitable detectable molecules include fluorescent proteins, luciferase,enzyme substrates, and radiolabels. The binding moiety may be directlylabelled with a detectable label or it may be indirectly labelled. Forexample, the binding moiety may be an unlabelled antibody which can bedetected by another antibody which is itself labelled. Alternatively,the second antibody may have bound to it biotin and binding of labelledstreptavidin to the biotin is used to indirectly label the firstantibody.

Chimeric Antigen Receptors

The present invention provides a chimeric antigen receptor (CAR) capableof binding to a mutant p53 polypeptide. The CAR comprises an antigenbinding fragment or polypeptide according to the present invention.

Chimeric Antigen Receptors (CARs) are recombinant receptors that provideboth antigen-binding and T cell activating functions. CAR structure andengineering is reviewed, for example, in Dotti et al., Immunol Rev(2014) 257(1), hereby incorporated by reference in its entirety.

Antigen-binding fragments according to the present invention areprovided herein as the antigen-binding domain of a chimeric antigenreceptor (CAR). In some embodiments, the CAR comprises a V_(L) domainand a V_(H) domain according to any embodiment of an antibody, antigenbinding fragment or polypeptide described herein. Accordingly, theantigen bound by the CAR according to the present invention is a mutantp53 polypeptide as described herein.

CARs may be combined with costimulatory ligands, chimeric costimulatoryreceptors or cytokines to further enhance T cell potency, specificityand safety (Sadelain et al., The basic principles of chimeric antigenreceptor (CAR) design. Cancer Discov. 2013 April; 3(4): 388-398.doi:10.1158/2159-8290.CD-12-0548, specifically incorporated herein byreference).

The present invention also provides a cell comprising a CAR according tothe invention. The CAR according to the present invention may be used togenerate tumor-targeted T cells, e.g. T cells targeted to tumor cellsexpressing the mutant p53 polypeptide for which the CAR is specific.

Engineering of CARs into T cells may be performed during culture, invitro, for transduction and expansion, such as happens during expansionof T cells for adoptive T cell therapy. The transduction may utilize avariety of methods, but stable gene transfer is required to enablesustained CAR expression in clonally expanding and persisting T cells.

In addition to the mutant p53 polypeptide specificity determiningelements described herein, CAR molecules may be further engineered toexpress co-stimulatory endodomains such as those derived from CD28 andtumor necrosis factor receptor superfamily member 9 (TNFRSF9; 4-1BB) topromote T cell proliferation and persistence upon encountering tumorcells (Nishio and Dotti, Oncolmmunology 4:2, e988098; February 2015).

A CAR typically combines an antigen binding domain with an intracellulardomain of the CD3-zeta chain or FcγRI protein in a single chimericprotein. The structural features of a CAR are described by Sjouke etal., (The pharmacology of second-generation chimeric antigen receptors.Nature Reviews Drug Discovery, 14, 499 509 (2015) doi:10.1038/nrd4597).A CAR typically has an extracellular antigen-binding domain linked to atransmembrane domain and endodomain. An optional hinge or spacer domainmay provide separation between the binding moiety and transmembranedomain and may act as a flexible linker.

In accordance with the present invention, the antigen recognition domainof the CAR is, or is derived from, an antibody, antigen binding fragmentor polypeptide which is capable of binding to a mutant p53 polypeptide,as described herein.

Hinge or spacer regions may be flexible domains allowing the bindingmoiety to orient in different directions. Hinge or spacer regions may bederived from IgG1 or the CH₂CH₃ region of immunoglobulin.

Transmembrane domains may be hydrophobic alpha helix that spans the cellmembrane. The transmembrane domain associated with the endodomain iscommonly used.

The endodomain is responsible for receptor clustering/dimerization afterantigen binding and for initiation of signal transduction to the cell.One commonly used transmembrane domain is the CD3-zeta transmembrane andendodomain. Intracellular domains from one or more co-stimulatoryprotein receptors, such as CD28 4-1 BB, OX40, ICOS, may optionally beincorporated into the cytoplasmic tail of the CAR to provide additionalco-stimulatory signaling, which may be beneficial in terms of anti-tumoractivity.

In one embodiment, a CAR comprises an extracellular domain having anantigen recognition domain, a transmembrane domain, and a cytoplasmicdomain. A transmembrane domain that is naturally associated with one ofthe domains in the CAR may be used or the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins to minimize interactions with other members of thereceptor complex. The cytoplasmic domain may be designed to comprise theCD28 and/or 4-1 BB signaling domain by itself or be combined with anyother desired cytoplasmic domain(s). The cytoplasmic domain may bedesigned to further comprise the signaling domain of CD3-zeta. Forexample, the cytoplasmic domain of the CAR can include but is notlimited to CD3-zeta, 4-1 BB and CD28 signaling modules and combinationsthereof.

The present invention also provides CAR T cells comprising as a CAR anantigen binding fragment capable of binding to a mutant p53 polypeptide,according to the present invention.

CAR T cells of the invention can be generated by introducing alentiviral vector in vitro comprising a desired CAR, for example a CARcomprising anti-mutant p53, CD8a hinge and transmembrane domain, andhuman 4-1BB and CD3zeta signaling domains, into the cells.

The CAR T cells of the invention are able to replicate in vivo resultingin long-term persistence that can lead to sustained tumor control.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR capable of binding to a mutant p53polypeptide for the treatment of a patient having cancer or at risk ofhaving cancer using lymphocyte infusion. Preferably, autologouslymphocyte infusion is used in the treatment. Autologous PBMCs arecollected from a patient in need of treatment and T cells are activatedand expanded using the methods described herein and known in the art andthen infused back into the patient.

Methods for Detecting Mutant p53 Polypeptides

Antibodies, antibody fragments, polypeptides, conjugates, CARs or cellsdescribed herein may be used in methods that involve the binding of theantibody or antigen binding fragment to a mutant p53 polypeptide. Suchmethods may involve detection of the bound complex of antibody, antibodyfragment, polypeptide, conjugate, CAR or cell, and mutant p53polypeptide. As such, in one embodiment a method is provided, the methodcomprising contacting a sample containing, or suspected to contain, amutant p53 polypeptide with an antibody, antibody fragment, polypeptide,conjugate, CAR or cell as described herein and detecting the formationof a complex of antibody, antibody fragment, polypeptide, conjugate, CARor cell, and mutant p53 polypeptide.

In aspects of the present invention, an in vitro complex is provided,comprising an antibody, antibody fragment, polypeptide, conjugate, CARor cell according to the invention bound to (i.e. in complex with) themutant p53 polypeptide (or a fragment thereof comprising the mutation)for which the antibody, antibody fragment, polypeptide, conjugate, CARor cell is specific.

Suitable method formats are well known in the art, includingimmunoassays such as sandwich assays, e.g. ELISA. The method may involvelabelling the antibody, antibody fragment, polypeptide, conjugate, CARor cell, or mutant p53 polypeptide, or both, with a detectable label,e.g. fluorescent, luminescent or radio-label. Expression of a mutant p53polypeptide may be measured by immunohistochemistry (IHC), for exampleof a tissue sample obtained by biopsy.

In particular, the anti-mutant p53 antibodies of the present inventionare useful in analysis by immunoblot, immunofluorescence,immunoprecipitation, immunohistochemistry and in vivo imaging, asexemplified herein.

Methods of this kind may provide the basis of a method of diagnosis orprognostic evaluation of a disease or condition requiring detectionand/or quantitation of a mutant p53 polypeptide. Such methods may beperformed in vitro on a patient sample, or following processing of apatient sample. Once the sample is collected, the patient is notrequired to be present for the in vitro method of diagnosis orprognostic evaluation to be performed and therefore the method may beone which is not practised on the human or animal body.

The methods may involve detecting the presence of a mutant p53polypeptide present in a patient sample.

The methods may involve determining the amount of a mutant p53polypeptide present in a patient sample. In some embodiments the methodsmay further comprise comparing the determined amount against a standardor reference value as part of the process of reaching a diagnosis. Otherdiagnostic tests may be used in conjunction with those described here toenhance the accuracy of the diagnosis or prognosis or to confirm aresult obtained by using the tests described here.

Detection in a sample of a mutant p53 polypeptide may be used for thepurpose of diagnosis of a cancerous condition in the patient, diagnosisof a predisposition to a cancerous condition or for providing aprognosis (prognosticating) of a cancerous condition. The diagnosis orprognosis may relate to an existing (previously diagnosed) cancerouscondition, which may be benign or malignant, may relate to a suspectedcancerous condition or may relate to the screening for cancerousconditions in the patient (which may be previously undiagnosed).

Detection in a sample of a mutant p53 polypeptide may be indicative thata patient may respond to treatment with an anti-mutant p53 antibody,fragment, polypeptide, conjugate, CAR or cell. The presence of a givenmutant p53 polypeptide in a sample may be used to select a patient fortreatment with a given anti-mutant p53 antibody, fragment, polypeptide,conjugate, CAR or cell. The antibodies, antibody fragments,polypeptides, conjugates, CARs or cells of the present invention maytherefore be used to select a patient for treatment with anti-mutant p53antibody therapy.

A sample may be taken from any tissue or bodily fluid. The sample maycomprise or may be derived from: a quantity of blood; a quantity ofserum derived from the individual's blood which may comprise the fluidportion of the blood obtained after removal of the fibrin clot and bloodcells; a tissue sample or biopsy; or cells isolated from saidindividual.

Methods according to the present invention are preferably performed invitro. The term “in vitro” is intended to encompass experiments withcells in culture whereas the term “in vivo” is intended to encompassexperiments with intact multi-cellular organisms.

The invention also provides the antibody, antibody fragment,polypeptide, conjugate, CAR or cell according to the invention for usein methods for detecting, localizing or imaging a cancer (e.g. atumour), e.g. in vivo.

The antibody, fragment, polypeptide, conjugate, CAR or cell according tothe invention may be suitably labeled directly or indirectly with adetectable label (e.g. a signal-generating label), such as a radioactiveisotope or non-isotopic entity, for detection. Radioisotopes includelodine¹²³, Iodine¹²⁵, Iodine¹²⁶, Iodine¹³¹, Iodine¹³³, Bromine⁷⁷,Technetium^(99m), Indium¹¹¹, Indium^(113m), Gallium⁶⁷, Gallium⁶⁸,Ruthenium⁹⁵, Ruthenium⁹⁷, Ruthenium¹⁰³, Ruthenium¹⁰⁵, Mercury²⁰⁷,Mercury²⁰³, Rhenium^(99m), Rhenium¹⁰¹, Rhenium¹⁰⁵, Scandium⁴⁷,Tellurium^(121m), Tellurium^(122m), Tellurium^(125m), Thulium¹⁶⁵,Thulium¹⁶⁷, Thulium¹⁶⁸, Copper⁶⁷, Fluorine¹⁸, Yttrium⁹⁰, Palladium¹⁰⁰,Bismuth²¹⁷ and Antimony²¹¹. Nonisotopic entities may be selected fromenzymes (e.g. peroxidase, alkaline phosphatase, glucose oxidase,beta-galactosidase, luciferase), dyes, haptens, luminescent agents suchas radioluminescent, chemiluminescent (e.g. acridinium ester, luminol,isoluminol), bioluminescent, fluorescent (e.g. fluorescein, rhodamine,eosine and NDB, green fluorescent protein (GFP) chelates of rare earthssuch as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin,Cy3, Cy5) or phosphorescent agents, antibodies, receptors and ligandssuch as biotin, avidin, streptavidin or digoxigenin.

Detection techniques are well known to those of skill in the art and canbe selected to correspond with the labelling agent. Suitable techniquesinclude PCR amplification of oligonucleotide tags, mass spectrometry,detection of fluorescence or colour, e.g. upon enzymatic conversion of asubstrate by a reporter protein, or detection of radioactivity.

In some embodiments, the methods comprising administering the antibody,antibody fragment, polypeptide, conjugate, CAR or cell to a subject,e.g. a patient diagnosed with or suspected of having a cancer anddetecting the antibody, antibody fragment, polypeptide, conjugate, CARor cell. In some embodiments, the methods comprise detecting signal fromthe antibody, antibody fragment, polypeptide, conjugate, CAR or cell. Insome embodiments the method comprises conversion of the signal to animage.

The present invention also provides methods for selecting/stratifying asubject for treatment with a mutant p53 polypeptide-targeted agent usingthe antibody, fragment, polypeptide, conjugate, CAR, or cell accordingto the invention. In some embodiments, a subject is selected fortreatment in accordance with the invention, or is identified as asubject which would benefit from such treatment, based on detection ofthe presence of the mutant p53 polypeptide, or nucleic acid encoding themutant p53 polypeptide, e.g. in a sample obtained from the individual.

Therapeutic Applications

Antibodies, antigen binding fragments, polypeptides, conjugates, CARsand cells according to the present invention and compositions comprisingsuch agents may be provided for use in methods of medical treatment.Treatment may be provided to subjects having a disease or condition inneed of treatment. The disease or condition may be a cancer.

In some embodiments, the cancer may comprise cells which express amutant p53 polypeptide. In some embodiments, the cancer may comprisecells expressing the mutant p53 polypeptide for which the anti-mutantp53 antibody, fragment or polypeptide according to the invention isspecific.

For example, such tumor cells expressing mutant p53 polypeptide may bekilled directly by treatment with antibodies according to the presentinvention, by antibody dependent cell-mediated cytotoxicity (ADCC),complement dependent cytotoxicity (CDC), or using antibody-drugconjugates.

The treatment may be aimed at prevention of the development orprogression of a cancer. As such, the antibodies, antigen bindingfragments, polypeptides or conjugates may be used to formulatepharmaceutical compositions or medicaments and subjects may beprophylactically treated against development of a disease state. Thismay take place before the onset of symptoms of the disease state, and/ormay be given to subjects considered to be at greater risk of developmentor progression of cancer.

Administration of an antibody, antigen binding fragment or polypeptideis preferably in a “therapeutically effective amount”, this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease being treated. Prescription oftreatment, e.g. decisions on dosage etc., is within the responsibilityof general practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Antibody Conjugates

The present invention also provides antibody conjugates, comprising anantibody, antigen binding fragment or polypeptide according to theinvention conjugated to a chemical moiety.

In some embodiments, the chemical moiety may be a moiety for providing atherapeutic effect. In some embodiments, the chemical moiety may be adrug moiety (e.g. a cytotoxic agent). In some embodiments, the drugmoiety may be a chemotherapeutic drug as described hereinbelow.

In some embodiments the chemical moiety may be a detectable moiety.Suitable moieties and means for their detection are well known to thosein the art and include the radioactive isotope or non-isotopic entitiesdescribed hereinabove.

Formulating Pharmaceutically Useful Compositions and Medicaments

Antibodies, antigen binding fragments, polypeptides, conjugates, CARsand cells according to the present invention may be formulated aspharmaceutical compositions for clinical use and may comprise apharmaceutically acceptable carrier, diluent, excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating anantibody, antigen binding fragment, polypeptide, CAR, or cell asdescribed herein; and/or mixing an isolated antibody, antigen bindingfragment, polypeptide, CAR, or cell as described herein with apharmaceutically acceptable carrier, adjuvant, excipient or diluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a medicament or pharmaceuticalcomposition for use in the treatment of a cancer, the method comprisingformulating a pharmaceutical composition or medicament by mixing anantibody, antigen binding fragment, polypeptide, CAR, or cell asdescribed herein with a pharmaceutically acceptable carrier, adjuvant,excipient or diluent.

Compositions may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

In this specification an antibody, antigen binding fragment,polypeptide, CAR, or cell of the present invention and achemotherapeutic agent may be administered simultaneously orsequentially.

In some embodiments, treatment with an antibody, antigen bindingfragment, polypeptide, CAR, or cell of the present invention may beaccompanied by chemotherapy.

Simultaneous administration refers to administration of the antibody,antigen binding fragment, polypeptide, CAR, or cell and therapeuticagent together, for example as a pharmaceutical composition containingboth agents (combined preparation), or immediately after each other andoptionally via the same route of administration, e.g. to the sameartery, vein or other blood vessel.

Sequential administration refers to administration of one of theantibody, antigen binding fragment, polypeptide, CAR, or cell ortherapeutic agent followed after a given time interval by separateadministration of the other agent. It is not required that the twoagents are administered by the same route, although this is the case insome embodiments. The time interval may be any time interval.

Cancer

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, salivary gland, sigmoid colon,skin, small intestine, soft tissues, spleen, stomach, testis, thymus,thyroid gland, tongue, tonsil, trachea, uterus, vulva, white bloodcells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma. In some embodiments, the cancer may be breast cancer.

In some embodiments the cancer is a cancer encoding or expressing amutant p53 peptide or polypeptide. In some embodiments the cancer is acancer encoding or expressing a mutant p53 peptide/polypeptidecomprising a mutation in the DNA-binding domain (DBD).

In some embodiments, the cancer is a cancer encoding or expressing amutant p53 peptide/polypeptide comprising an amino acid differencerelative to the wildtype p53 sequence at one or more of positions 248,273, 175, 176, 179, 220, 245, 249, 282 or 337.

In some embodiments, the cancer is a cancer encoding or expressing amutant p53 peptide/polypeptide comprising one or more of the followingmutations: R175H, R2480, R273H, R248W, G245S, R273C, R282W, R249S,G245D, C176F, H179Y, H179R, Y220C, and R337H.

A cancer may be determined to encode or express a mutant p53peptide/polypeptide by any suitable means, which are well known to theskilled person, e.g. based on analysis of a biological sample.

A cancer encoding a mutant p53 polypeptide may be identified on thebasis of detection of nucleic acid encoding the mutation, e.g. by DNAsequencing etc. A cancer expressing a mutant p53 polypeptide may beidentified by detection of expression of a mutant p53peptide/polypeptide. Expression may be gene expression or proteinexpression. Gene expression can be determined e.g. by detection of mRNAencoding a mutant p53 peptide/polypeptide, for example by quantitativereal-time PCR (qRT-PCR). Protein expression can be determined e.g. bydetection of mutant p53 peptide/polypeptide, for example byantibody-based methods, for example by western blot,immunohistochemistry, immunocytochemistry, flow cytometry, ELISA.

Herein, “a cancer encoding or expressing a mutant p53peptide/polypeptide” includes any cell encoding or expressing a mutantp53 peptide/polypeptide. In some embodiments, the cell may be a cell ofa tumor.

Chemotherapy/Radiotherapy

Chemotherapy and radiotherapy respectively refer to treatment of acancer with a drug or with ionising radiation (e.g. radiotherapy usingX-rays or γ-rays).

The drug may be a chemical entity, e.g. small molecule pharmaceutical,antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor),or a biological agent, e.g. antibody, antibody fragment, nucleic acid orpeptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, orprotein. The drug may be formulated as a pharmaceutical composition ormedicament. The formulation may comprise one or more drugs (e.g. one ormore active agents) together with one or more pharmaceuticallyacceptable diluents, excipients or carriers.

A treatment may involve administration of more than one drug. A drug maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. For example, the chemotherapy may be a co-therapy involvingadministration of two drugs, one or more of which may be intended totreat the cancer.

The chemotherapy may be administered by one or more routes ofadministration, e.g. parenteral, intravenous injection, oral,subcutaneous, intradermal or intratumoral.

The chemotherapy may be administered according to a treatment regime.The treatment regime may be a pre-determined timetable, plan, scheme orschedule of chemotherapy administration which may be prepared by aphysician or medical practitioner and may be tailored to suit thepatient requiring treatment.

The treatment regime may indicate one or more of: the type ofchemotherapy to administer to the patient; the dose of each drug orradiation; the time interval between administrations; the length of eachtreatment; the number and nature of any treatment holidays, if any etc.For a co-therapy a single treatment regime may be provided whichindicates how each drug is to be administered.

Chemotherapeutic drugs and biologics may be selected from: alkylatingagents such as cisplatin, carboplatin, mechlorethamine,cyclophosphamide, chlorambucil, ifosfamide; purine or pyrimidineanti-metabolites such as azathiopurine or mercaptopurine; alkaloids andterpenoids, such as vinca alkaloids (e.g. vincristine, vinblastine,vinorelbine, vindesine), podophyllotoxin, etoposide, teniposide, taxanessuch as paclitaxel (Taxol™), docetaxel;

topoisomerase inhibitors such as the type I topoisomerase inhibitorscamptothecins irinotecan and topotecan, or the type II topoisomeraseinhibitors amsacrine, etoposide, etoposide phosphate, teniposide;antitumor antibiotics (e.g. anthracyline antibiotics) such asdactinomycin, doxorubicin (Adriamycin™), epirubicin, bleomycin,rapamycin; antibody based agents, such as anti-PD-1 antibodies,anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-CTLA-4, anti-4-1 BB,anti-GITR, anti-CD27, anti-BLTA, anti-OX43, anti-VEGF, anti-TNFα,anti-IL-2, antiGpIlb/IIIa, anti-CD-52, anti-CD20, anti-RSV,anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies,monoclonal antibodies or antibody fragments, examples include:cetuximab, panitumumab, infliximab, basiliximab, bevacizumab (Avastin®),abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (Mabthera®),palivizumab, trastuzumab, etanercept, adalimumab, nimotuzumab; EGFRinihibitors such as erlotinib, cetuximab and gefitinib; anti-angiogenicagents such as bevacizumab (Avastin®); cancer vaccines such asSipuleucel-T (Provenge®).

Further chemotherapeutic drugs may be selected from: 13-cis-RetinoicAcid, 2-Chlorodeoxyadenosine, 5-Azacitidine 5-Fluorouracil,6-Mercaptopurine, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-DAdriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin,Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®,Arsenic Trioxide, Asparaginase, ATRA Avastin®, Azacitidine, BCG, BCNU,Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU,Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, CalciumLeucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU,Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®,Cytarabine Cytosar-U®, Cytoxan®, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™ DTIC, DTIC-Dome®, Duralone®, Eligard™, Ellence™, Eloxatin™,Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, ErwiniaL-asparaginase, Estramustine, Ethyol Etopophos®, Etoposide, EtoposidePhosphate, Eulexin®, Everolimus, Evista®, Exemestane, Faslodex®,Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluoxymesterone, Flutamide, Folinic Acid, FUDR®,Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gleevec™,Gliadel® Wafer, Goserelin, Granulocyte-Colony Stimulating Factor,Granulocyte Macrophage Colony Stimulating Factor, Herceptin®, Hexadrol,Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, HydrocortAcetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate,Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea,Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®,IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase, Lanacort®,Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin,Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, LiposomalAra-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, LupronDepot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin® Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®.

Routes of Administration

Antibodies, antigen binding fragments, polypeptides, conjugates, CARs,cells and other therapeutic agents, medicaments and pharmaceuticalcompositions according to aspects of the present invention may beformulated for administration by a number of routes, including but notlimited to, parenteral, intravenous, intra-arterial, intramuscular,subcutaneous, intradermal, intratumoral and oral. Antibodies, antigenbinding fragments, polypeptides, conjugates, CARs, cells and therapeuticagents may be formulated in fluid or solid form. Fluid formulations maybe formulated for administration by injection to a selected region ofthe human or animal body.

Dosage Regimes

Multiple doses of the antibody, antigen binding fragment, polypeptide,conjugate, CAR or cell may be provided. One or more, or each, of thedoses may be accompanied by simultaneous or sequential administration ofanother therapeutic agent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

Kits

In some aspects of the present invention a kit of parts is provided. Insome embodiments the kit may have at least one container having apredetermined quantity of the antibody, antigen binding fragment,polypeptide, conjugate, CAR or cell according to the invention. The kitmay provide the antibody, antigen binding fragment, polypeptide,conjugate, CAR or cell in the form of a medicament or pharmaceuticalcomposition, and may be provided together with instructions foradministration to a patient in order to treat a specified disease orcondition. The antibody, antigen binding fragment or polypeptide may beformulated so as to be suitable for injection or infusion to a tumor orto the blood.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g.chemotherapeutic agent). In such embodiments, the kit may also comprisea second medicament or pharmaceutical composition such that the twomedicaments or pharmaceutical compositions may be administeredsimultaneously or separately such that they provide a combined treatmentfor the specific disease or condition. The therapeutic agent may also beformulated so as to be suitable for injection or infusion to a tumor orto the blood.

Subjects

The subject to be treated with an antibody, antigen binding fragment,polypeptide, conjugate, CAR or cell according to the invention may beany animal or human. The subject is preferably mammalian, morepreferably human. The subject may be a non-human mammal, but is morepreferably human. The subject may be male or female. The subject may bea patient. A subject may have been diagnosed with a disease or conditionrequiring treatment, or be suspected of having such a disease orcondition.

Sequence Identity

Alignment for purposes of determining percent amino acid or nucleotidesequence identity can be achieved in various ways known to a person ofskill in the art, for instance, using publicly available computersoftware such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software.When using such software, the default parameters, e.g. for gap penaltyand extension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNAGAPDIST=4.

Recombinant Production

The immunogen used in the methods according to the invention and theantibodies, fragments, polypeptides, conjugates and CARs according tothe invention may be prepared according to methods for recombinantproduction known to the skilled person.

The peptide/polypeptide of interest may be prepared by chemicalsynthesis, e.g. liquid or solid phase synthesis. For example,peptides/polypeptides can by synthesised using the methods described in,for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which ishereby incorporated by reference in its entirety.

The immunogens, antibodies, fragments, polypeptides, conjugates and CARsaccording the invention may be produced by recombinant expression.Molecular biology techniques suitable for recombinant production arewell known in the art, such as those set out in Green and Sambrook,Molecular Cloning: A Laboratory Manual (4^(th) Edition), Cold SpringHarbor Press, 2012, which is hereby incorporated by reference in itsentirety.

Expression may be from a nucleotide sequence. The nucleotide sequencemay be contained in a vector. A “vector” as used herein is anoligonucleotide molecule (DNA or RNA) used as a vehicle to transferforeign genetic material into a cell. The vector may be an expressionvector for expression of the foreign genetic material in the cell. Suchvectors may include a promoter sequence operably linked to thenucleotide sequence encoding the sequence to be expressed. A vector mayalso include a termination codon and expression enhancers. Any suitablevectors, promoters, enhancers and termination codons known in the artmay be used to express a peptide or polypeptide from a vector accordingto the invention. In some embodiments, the vector may be a plasmid, MAC,virus, etc. In some embodiments, the vector may be a eukaryoticexpression vector, e.g. a vector comprising the elements necessary forexpression of protein from the vector in a eukaryotic cell. In someembodiments, the vector may be a mammalian expression vector, e.g.comprising a cytomegalovirus (CMV) or SV40 promoter to drive proteinexpression.

The term “operably linked” may include the situation where a selectednucleotide sequence and regulatory nucleotide sequence (e.g. promoterand/or enhancer) are covalently linked in such a way as to place theexpression of the nucleotide sequence under the influence or control ofthe regulatory sequence (thereby forming an expression cassette). Thus aregulatory sequence is operably linked to the selected nucleotidesequence if the regulatory sequence is capable of effectingtranscription of the nucleotide sequence. The resulting transcript maythen be translated into a desired peptide or polypeptide.

For recombinant production according to the invention, any cell suitablefor the expression of polypeptides may be used. The cell may be aprokaryote or eukaryote. In some embodiments the cell is a prokaryoticcell, such as a cell of archaea or bacteria. In some embodiments thebacteria may be Gram-negative bacteria such as bacteria of the familyEnterobacteriaceae, for example Escherichia coli.

In some embodiments, the cell is a eukaryotic cell such as a yeast cell,a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK, HeLa orCOS cells.

In some cases the cell is not a prokaryotic cell because someprokaryotic cells do not allow for the same folding orpost-translational modifications as eukaryotic cells. In addition, veryhigh expression levels are possible in eukaryotes and proteins can beeasier to purify from eukaryotes using appropriate tags. Specificplasmids may also be utilised which enhance secretion of the proteininto the media.

Production may involve culture or fermentation of a eukaryotic cellmodified to express the peptide or polypeptide. The culture orfermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted peptide orpolypeptide. Culture, fermentation and separation techniques are wellknown to those of skill in the art, and are described, for example, inGreen and Sambrook, Molecular Cloning: A Laboratory Manual (4^(th)Edition; incorporated by reference herein above).

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culture of cells that express the immunogen, antibody,fragment, polypeptide, conjugate or CAR, the peptide/polypeptide ofinterest is preferably isolated. Any suitable method for separatingproteins from cell culture known in the art may be used. In order toisolate the peptide/polypeptide from a culture, it may be necessary tofirst separate the cultured cells from media containing thepeptide/polypeptide of interest. If the peptide/polypeptide of interestis secreted from the cells, the cells may be separated from the culturemedia that contains the secreted peptide/polypeptide of interest bycentrifugation. If the peptide/polypeptide of interest collects withinthe cell it will be necessary to disrupt the cells prior tocentrifugation, for example using sonification, rapid freeze-thaw orosmotic lysis. Centrifugation will produce a pellet containing thecultured cells, or cell debris of the cultured cells, and a supernatantcontaining culture medium and the peptide/polypeptide of interest.

It may then be desirable to isolate the peptide/polypeptide of interestfrom the supernatant or culture medium, which may contain other proteinand non-protein components. A common approach to separating proteincomponents from a supernatant or culture medium is by precipitation.Proteins of different solubilities are precipitated at differentconcentrations of precipitating agent such as ammonium sulfate. Forexample, at low concentrations of precipitating agent, water solubleproteins are extracted. Thus, by adding different increasingconcentrations of precipitating agent, proteins of differentsolubilities may be distinguished. Dialysis may be subsequently used toremove ammonium sulfate from the separated proteins.

Other methods for distinguishing different proteins are known in theart, for example ion exchange chromatography and size chromatography.These may be used as an alternative to precipitation, or may beperformed subsequently to precipitation.

Once the peptide/polypeptide of interest has been isolated from cultureit may be desired or necessary to concentrate the peptide orpolypeptide. A number of methods for concentrating proteins are known inthe art, such as ultrafiltration or lyophilisation.

Vaccination

The immunogens used to raise antibodies against mutant p53 polypeptidesin the methods of the present invention are also useful as vaccines. Theimmunogens can be used to generate immunity to cancer associated withmutation of p53.

Accordingly, the present invention provides a vaccine comprising,methods for vaccination using, and the use as a vaccine of, an immunogenin accordance with any embodiment as described herein.

The immunogen of the vaccine/used in vaccination may comprise a mutationof p53 for generating immunity to a cancer comprising the same mutation.For example, an immunogen comprising an antigen sequence comprising theR175H may be useful in a method for vaccination against a cancercomprising a cell or cells expressing a R175H p53, or comprising nucleicacid encoding a R175H p53.

In some embodiments multiple, different immunogens may be used in avaccine or vaccination according to the present invention. In someembodiments, vaccines/vaccination may use immunogens comprisingdifferent mutations of p53, and may therefore be useful to vaccinateagainst cancers comprising more than one p53 mutation and/or cancerscomprising different p53 mutations.

The skilled person is readily able to determine suitable formulationsfor vaccines and schedules for vaccination in accordance with thepresent invention, e.g. by reference to Vaccines (6^(th) Edn.) Plotkinet al. 2012, Elsevier Saunders, which is hereby incorporated byreference in its entirety.

Methods of vaccination and associated uses include prophylactic and/orpreventative treatment. For example, subjects considered to be at riskof developing a cancer, or at risk of recurrence or relapse followingtreatment, may be administered a vaccine as described herein. Suchadministration may occur whilst the subject is considered cancer free.Assessment of risk status and cancer status may be performed by asuitably qualified medical practitioner. Such methods may reduce thelikelihood of the cancer developing, growing, metastasising, orrecurring.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIGS. 1A to 1C. Amino acid sequences of immunogens used to raiseantibodies specific for p53 mutants. (1A) Sequence for immunogen used toraise antibodies against R175H p53 (SEQ ID NO:80). Three copies of themutant p53 sequences of variable lengths, each harbouring one ofmutation, were inserted into the active site of TrxA. (1B) Sequence forimmunogen used to raise antibodies against R248Q p53 (SEQ ID NO:84).(1C) Sequence for immunogen used to raise antibodies against R273H p53(SEQ ID NO:88). Amino acid sequences of mutant p53 are highlighted(light grey), and the mutant residue is indicated (dark grey). FlexibleGly/Ser linker sequences are underlined. The C-terminal 6-His tag usedfor purification is shown in italics.

FIGS. 2A to 2C. Predicted Swiss Models for the 3D structure of theimmunogens used to raise antibodies specific for p53 mutants. (2A)Predicted structure for immunogen used to raise antibodies against R175Hp53. (2B) Predicted structure for immunogen used to raise antibodiesagainst R248Q p53. (2C) Predicted structure for immunogen used to raiseantibodies against R273H p53. Structures corresponding to thethioredoxin and antigen sequences are indicated, as are the mutations.

FIGS. 3A to 3C. Photographs of SDS-PAGE (16%) profiles for total,soluble and insoluble proteins isolated from E. coli strain BL21 (DE3)expressing (3A) TrxR175H, (3B) TrxR248Q, and (3C) TrxR273H, before (0 h)and 4 h after IPTG induction. The expected expressed protein isindicated by the arrow. MW=Protein molecular weight ladder (PrecisionPlus Protein™ All Blue Standards, Bio-Rad).

FIGS. 4A and 4B. Mass spectra for purified (4A) TrxR175H and (4B)TrxR248Q, showing primary protein entity of the indicated molecularmasses.

FIGS. 5A to 5C. Graphs showing the results of ELISA screening of p53mutant specific antibodies. Cell culture supernatants obtained fromindividual hybridoma clones generated from spleens of mice immunizedwith (5A) TrxR175H, (5B) TrxR248Q, and (5C) TrxR273H were screened byELISA against the indicted whole proteins and peptide fragments.

FIGS. 6A to 6C. Bar charts showing the results of ELISA screening of p53mutant specific antibodies. Cell culture supernatants obtained fromindividual hybridoma clones generated from spleens of mice immunizedwith (6A) TrxR175H, (6B) TrxR248Q, and (6C) TrxR273H were screened byELISA against the indicted whole proteins and peptide fragments.

FIGS. 7A to 7C. Sequence alignments showing the results of phage displayexperiments for determining the epitope bound by the anti-mutant p53antibodies. Sequences for the peptides captured by cell culturesupernatant of hybridomas producing (7A) anti-R175H antibody (SEQ IDNOs:77, and 93-101) (7B) anti-R248Q antibody (SEQ ID NOs:102-110) and(7C) anti-R273H antibody (SEQ ID NOs: 111-119). Consensus sequences areshown.

FIGS. 8A and 8B. Sequence alignments showing the results of phagedisplay experiments for determining the epitope bound by the anti-mutantp53 antibodies. Sequences for the peptides captured by cell culturesupernatant of the indicated hybridomas producing (8A) anti-R175Hantibody (SEQ ID NOs:77, 93, and 120-135) and (8B) anti-R248Q antibody(SEQ ID NOs:102, 103, and 136-144). Consensus sequences are shown.

FIG. 9A to 9D. Tables and Bar charts showing results of alanine scanELISAs for determining the epitope bound by anti-R175H antibody clones(9A) 4H5 (SEQ ID NOs:145-159), (9B) 7B9 (SEQ ID NOs:79, and 145-159) and(9C) 1008 (SEQ ID NOs:145-159), and (9D) anti-R273H antibody clone 13E4(SEQ ID NOs:160-173). Individually alanine substituted 13-mer peptidessurrounding the mutated hotspots (His 175 and His 273) were used todetermine the critical amino acid residues required for the mutantantibodies binding to mutant p53. Absorbance was measured at 650 nmafter 5 minutes' incubation with TMB substrate. Optical density readingsof individual alanine substituted peptides are shown. Critical residuesfor antibody binding are indicated motifs.

FIG. 10 . Sequence showing the epitopes for the antibodies identified byalanine scan analysis of the critical residues that the antibodiesrecognise (SEQ ID NO:174).

FIG. 11 . Photographs showing the results of immunoblots using mutantp53-specific antibodies. p53-null H1299 cells were transientlytransfected with plasmids encoding the indicated p53 mutant or wildtypep53 polypeptide, and protein lysates were used for direct immunoblottingwith the indicated antibodies. DO1 is a mouse mAb that detects all formsof human p53, and CM1 is a rabbit polyclonal antibody against human p53.

FIG. 12 . Photographs showing the results of immunoprecipitation assaysusing mutant p53-specific antibodies. p53-null H1299 cells weretransiently transfected with plasmids encoding the indicated p53 mutantor wildtype p53 polypeptide, and protein lysates were used forimmunoprecipitation assays. DO1 is a mouse mAb that detects all forms ofhuman p53, and CM1 is a rabbit polyclonal antibody against human p53.Pull-downs were performed with both the p53-mutant specific mAbsfollowed by immunoblot with CM1 antibody, and in the reverse order bypull down with CM1 and immunoblotting with the mutant-specific mAbs.

FIG. 13 . Table showing p53 mutation status of cell lines used foranalysis of p53-specific antibodies in FIGS. 14 and 15 .

FIG. 14 . Photographs showing the results of immunoblots using mutantp53-specific antibodies. Protein lysates of the indicated cell lineswere used for direct immunoblotting with the indicated antibodies. A549and MCF-7 are wild-type p53 carrying cell lines that were used withoutor with UV irradiation to induce the endogenous p53 expression.

FIG. 15 . Photographs showing the results of immunoprecipitation assaysusing mutant p53-specific antibodies. Protein lysates of the indicatedcell lines were used for immunoprecipitation assays using the indicatedantibodies. A549 and MCF-7 are wild-type p53 carrying cell lines thatwere used without or with UV irradiation to induce the endogenous p53expression. Pull-downs were performed with both the p53-mutant specificmAbs followed by immunoblot with CM1 antibody, and in the reverse orderby pull down with CM1 and immunoblotting with the mutant-specific mAbs.

FIG. 16 . Photographs showing the results of immunofluorescenceanalyses. p53-null H1299 cells were transiently transfected withplasmids encoding the indicated p53 mutant or wildtype p53 polypeptideand subjected to immunofluorescence analysis.

FIGS. 17A and 17B. Photographs showing the results of immunofluorescenceanalyses. (17A) The indicated cell lines were used forimmunofluorescence analysis using the indicated antibodies. (17B) R248QmAb in p53-null H1299 cells expressing either the R248Q or R248W mutantp53 polypeptide. Dapi staining highlights nuclei.

FIGS. 18A and 18B. Photographs relating to immunohistochemical analysisusing mutant-specific anti-p53 antibodies. (18A) Sequence analysis ofhuman colon tumour samples to determined mutational status for p53 (SEQID NOs:175-186), and (18B) staining of the paraffin-embedded tumoursamples using the indicated anti-p53 antibodies.

FIG. 19 . Photographs showing the results of human tumour microarraysusing R175H mutant-specific anti-p53 antibodies. Human tumor microarraysfrom triple negative breast cancer, colorectal cancers, renal cancersand lung cancers were stained with R175H mutant p53-specific mAb and thepan-p53 mAb DO7. Double positive refers to samples that were stained byboth DO7 and the mutation-specific mAb, and single positives were onlystained by DO7. There were no samples with only mutation-specific mAbstaining without DO7 staining.

FIGS. 20A and 20B. Chromatograms showing p53 sequence in tumors samples,and staining for anti-p53 antibodies and anti-R175H antibody byimmunohistochemistry. Human Triple negative breast cancer samples werestained with anti-p53 (DO7 and 11 D1) antibodies and the anti-R175H p53antibody. (20A) Samples B41, B89 and B98 showing positive staining bythe R175H antibody, as well as both anti-p53 antibodies DO7 and 11 D1.Sequences show that the samples comprise the R175H mutation(CAG—underlined) (SEQ ID NOs:187-189). (20B) Sample B27 showing positivestaining only by the anti-p53 antibodies DO7 and 11 D1 (and not by theanti-R175H antibody), and sample B52, not stained by any of the anti-p53antibodies. Sequences show that B27 and B57 do not comprise a mutationat position 175 (CGC—underlined) (SEQ ID NOs:190 and 191).

FIG. 21 . Table summarising the results of human tumour microarrayanalysis by immunohistochemistry.

FIG. 22 . Amino-acid sequence alignments of p53 from various species areshown, with regions containing the R175 (SEQ ID NOs:192-214), R248 orR273 (SEQ ID NOs:215-235) residues (indicated by arrows) and thesurrounding residues underlined, which shows strong conservation crossspecies, indicating that the mutant-specific mAbs could work across allspecies.

FIGS. 23A to 23D. Photographs showing the results of analysis of themouse equivalent to the R175H mutation (i.e. R172H). Mouse embryonicfibroblasts from the indicated genotypes were used for (23A) directimmunoblotting (I.B.) or (23B) immunoprecipitation (I.P.), (23C)immunofluorescence, or (23D) Immunohistochemical analysis with theanti-R175H-specific mAb. Polyclonal antibody against mouse p53, CM5, wasused for I.B. in immunoprecipitation studies. R246S mutant MEFs are froma different hot-spot mutant knock-in mouse strain.

FIG. 24 . Light chain variable domain sequences for anti-R175H p53antibody clones 4H5, 7B9 and 1008. CDRs are underlined and shownseparately.

FIG. 25 . Heavy chain variable domain sequences for anti-R175H p53antibody clones 4H5, 7B9 and 1008. CDRs are underlined and shownseparately.

FIG. 26 . Table summarising light and heavy chain CDRs for anti-R175Hp53 antibody clones 4H5, 7B9 and 1008, and indicating consensussequences.

FIG. 27 . Nucleotide and encoded amino acid sequences of heavy and lightchain variable domain sequences for anti-R175H p53 antibody clones 4H5,7B9 and 1008.

FIG. 28 . Light chain variable domain sequences for anti-R248Q p53antibody clones 3G11 and 4H2. CDRs are underlined and shown separately.

FIG. 29 . Heavy chain variable domain sequences for anti-R248Q p53antibody clones 3G11 and 4H2. CDRs are underlined and shown separately.

FIG. 30 . Table summarising light and heavy chain CDRs for anti-R248Qp53 antibody clones 3G11 and 4H2, and indicating consensus sequences.

FIG. 31 . Nucleotide and encoded amino acid sequences of heavy and lightchain variable domain sequences for anti-R248Q p53 antibody clones 3G11and 4H2.

FIG. 32 . Light chain variable domain sequence for anti-R273H p53antibody clone 13E4. CDRs are underlined and shown separately.

FIG. 33 . Heavy chain variable domain sequence for anti-R273H p53antibody clone 13E4. CDRs are underlined and shown separately.

FIG. 34 . Nucleotide and encoded amino acid sequences of heavy and lightchain variable domain sequences for anti-R273H p53 antibody clone 13E4.

FIG. 35 . Photographs showing the results of immunoblots of extractsfrom R172H mouse thymic lymphoma primary cell line, T47D, WiDr and DKOcells using 4H5, 7B9 and 1008 hybridoma cell culture supernatant.

FIGS. 36A to 36C. Photographs showing the results Immunofluorescenceanalysis of cells expressing and non-expressing R175H mutant p53proteins with hybridoma cell culture supernatant from clones (36A) 4H5(36B) 7B9 and (36C) 10C8.

FIG. 37 . Photographs showing the results of analysis of mouseintestinal tissue sections (p53 knockout or irradiated, p53 R172Hpositive) using hybridoma cell culture supernatant from clones 4H5, 7B9and 1008.

FIG. 38 . Photographs showing tumor targeting by the XenoLight CF750conjugated 4H5 mAb. Mice were imaged at 6, 24, 48, 72 hours and 7 daysafter i.v. injection of 100 ug XenoLight CF750 labelled 4H5 mAb andimaged by IVIS. Mouse tumor cells derived from P53^(R172H/R172H) micewere transfected with luciferase gene and used to establish the tumormodel.

FIG. 39 . Photograph showing the results of immunoblots of extracts fromTKO and HCC70 cells using 3G11 and 4H2 hybridoma cell culturesupernatant.

FIGS. 40A and 40B. Photographs showing the results Immunofluorescenceanalysis of cells expressing R248Q p53, and cells not expressing R248Qp53, with hybridoma cell culture supernatant from clones (40A) 3G11(40B) 4H2.

FIG. 41 . Photograph showing the results of immunoblots of extracts fromT47D and MB468 cells using 13E4 hybridoma cell culture supernatant.

FIG. 42 . Photographs showing the results Immunofluorescence analysis ofcells expressing R273H p53 (WiDr), and cells not expressing R273H p53(T47D), with hybridoma cell culture supernatant from clone 13E4.

FIG. 43 . Photographs showing tumor targeting by the XenoLight CF750conjugated 13E4 mAb. Mice were imaged at 72 hours after i.v. injectionof 100 ug XenoLight CF750 labelled 13E4 mAb and imaged by IVIS. Thep53-R273H mutant HT29 tumor cell line transfected with luciferase gene,was used to establish the tumor model.

FIG. 44 . Photographs showing the results of analysis of mouseintestinal tissue sections (p53 knockout or irradiated, p53 R172Hpositive) using purified, mouse FV-human IgG1 chimeric 4H5, 7B9 and 1008antibodies.

FIG. 45 . Graph showing the results of analysis of anti-R175H antibodyto control growth of R175H-positive cancer in vivo. SKBR-3 cellscarrying luciferase were used to establish a human xenograft cancermodel, and the influence on tumor growth of treatment with anti-R175Hantibody (a175; dotted line) was compared to IgG control (IgG, solidline).

FIG. 46A to 46E. Graphs showing results of ELISA analysis of antibodyresponse in mice injected with immunogens corresponding to (46A and 46B)R175H, (46C and 46D) R273H or (46E and 46F) R248Q mutants of p53, topeptide corresponding to the p53 mutant or wildtype human p53. Serumfrom immunized mice was analysed for antibody capable of binding topeptide corresponding to the p53 mutant (46A) R175H, (46C) R273H, or(46E) R248Q, and full-length wildtype human p53 (WT HP53; 46B, 46D and46E).

FIGS. 47A to 47C. Photographs showing results of immunofluorescenceanalysis of ability of antibodies produced in response to immunisationwith immunogens corresponding to (47A) R175H, (47B) R248Q or (47C) R273Hmutants of p53 to recognise cells expressing the corresponding mutantp53 polypeptide.

FIG. 48 . Graph showing the effect of anti-R175H antibody on growth ofSKBR cells. Mice were injected with 5×10⁶ SKBR luciferase reportercells, and injected at regular intervals (injections are indicated byarrows) with 100 μl of monoclonal antibody specific for the R175H mutantp53 (a175) or isotype control antibody (IgG). SKBR cell growth wasmeasured by analysis of luciferase activity.

FIG. 49 . Light chain variable domain sequence for mouse human chimericanti-R175H p53 antibody clones MH 4H5, MH 7B9 and MH 10C8. CDRs areunderlined and shown separately.

FIG. 50 . Heavy chain variable domain sequence for mouse human chimericanti-R175H p53 antibody clones MH 4H5, MH 7B9 and MH 10C8. CDRs areunderlined and shown separately.

FIG. 51 . Nucleotide and encoded amino acid sequences of light chainvariable domain sequences for mouse human chimeric anti-R175H p53antibody clones MH 4H5, MH 7B9 and MH 10C8.

FIG. 52 . Nucleotide and encoded amino acid sequences of heavy chainvariable domain sequences for mouse human chimeric anti-R175H p53antibody clones MH 4H5, MH 7B9 and MH 10C8.

FIG. 53 . Light chain variable domain sequence for mouse human chimericanti-R273H p53 antibody clone MH 13E4. CDRs are underlined and shownseparately.

FIG. 54 . Heavy chain variable domain sequence for mouse human chimericanti-R273H p53 antibody clone MH 13E4. CDRs are underlined and shownseparately.

FIG. 55 . Nucleotide and encoded amino acid sequences of light chainvariable domain sequences for mouse human chimeric anti-R273H p53antibody clone MH 13E4.

FIG. 56 . Nucleotide and encoded amino acid sequences of heavy chainvariable domain sequences for mouse human chimeric anti-R273H p53antibody clone MH 13E4.

FIG. 57 . Light chain variable domain sequence for mouse human chimericanti-R248Q p53 antibody clones MH 3G11 and MH 4H2. CDRs are underlinedand shown separately.

FIG. 58 . Heavy chain variable domain sequence for mouse human chimericanti-R248Q p53 antibody clones MH 3G11 and MH 4H2. CDRs are underlinedand shown separately.

FIG. 59 . Nucleotide and encoded amino acid sequences of light chainvariable domain sequences for mouse human chimeric anti-R248Q p53antibody clones MH 3G11 and MH 4H2.

FIG. 60 . Nucleotide and encoded amino acid sequences of heavy chainvariable domain sequences for mouse human chimeric anti-R248Q p53antibody clones MH 3G11 and MH 4H2.

FIG. 61 . Bar chart showing the results of ELISA analysis confirmingspecific binding to R175H p53 by the chimeric mouse human antibodyclones MH 4H5, MH7B9 and MH 10C8. Averaged OD450 readings were plotted.The chimeric anti-p53 R175H antibodies (MH 4H5, MH 7B9, MH 10C8)antibodies produced positive signals between 0.1 and 1 ng/μlconcentrations. Data shown in this figure was for 1 ng/μl antibodyconcentration. All chimeric anti-p53 R175H antibodies specificallyrecognized and detected only human p53 R175H full length protein and nothuman p53 R273H full length, human p53 wild-type full length, or mousep53 wild-type full length protein (single asterisks). Commercialanti-p53 antibody (1c12) was included as a positive control antibody.(n=3).

FIG. 62 . Western blots showing specific binding to R175H p53 by thechimeric mouse human antibody clones MH 4H5, MH7B9 and MH 10C8. Humancell lines: H1299 (p53-null), MCF7, A549 (both wild-type p53), SKBR3(p53 R175H), A431, and SW480 (both p53 R273H) were tested in thisexperiment. All chimeric anti-p53 R175H antibodies recognized anddetected only endogenous human p53 R175H protein (SKBR3) and not humanp53 R273H, or human p53 wild-type protein (black arrow). All antibodieswere tested at 1 ng/μl concentration.

FIG. 63 . Representative images showing detection of endogenous p53proteins in human cell lines by MH 7B9. Cells were fixed in 4%paraformaldehyde then embedded in paraffin. Immunohistochemical assayswere performed using the following human cell lines: H1299 (p53-null),MCF7 (wild-type p53), SKBR3 (p53 R175H), and A431 (p53 R273H). Thephotographs show staining of all cell lines with 1.67 ng/μl of MH 7B9antibody. All chimeric anti-p53 R175H antibodies specifically recognizedand stained only cell nuclei expressing endogenous human p53 R175Hprotein (SKBR3 (top left panel)).

FIG. 64 . Bar chart showing the results of ELISA analysis confirmingspecific binding to R273H p53 by the chimeric mouse human antibody cloneMH 13E4. Averaged OD450 readings were plotted. Data shown in this figurewas for 1 ng/μl antibody concentration. MH 13E4 specifically recognizedand detected only human p53 R273H full length protein and not human p53R175H full length or human p53 wild-type full length protein (singleasterisks).

FIG. 65 . Western blot showing specific binding to R273H p53 by thechimeric mouse human antibody clone MH 13E4. Human cell lines: H1299(p53-null), MCF7, A549 (both wild-type p53), SKBR3 (p53 R175H), A431,and SW480 (both p53 R273H) were tested in this experiment. MH 13E4specifically recognized and detected only endogenous human p53 R273Hprotein (A431 and SW480) and not human p53 R175H, or human p53 wild-typeprotein (black arrow). MH 13E4 was tested at 1 ng/μl concentration.

FIG. 66 . Representative images showing detection of endogenous p53proteins in human cell lines by MH 13E4. Cells were fixed in 4%paraformaldehyde then embedded in paraffin. Immunohistochemical assayswere performed using the following human cell lines: H1299 (p53-null),MCF7 (wild-type p53), SKBR3 (p53 R175H), and A431 (p53 R273H). Thephotographs show staining of all cell lines with 5 ng/μl of MH 13E4antibody. MH 13E4 antibody specifically recognized and stained only cellnuclei expressing endogenous human p53 R273H protein (A431 (top rightpanel)).

FIG. 67 . Bar chart showing the results of ELISA analysis confirmingspecific binding to R248Q p53 by the chimeric mouse human antibodyclones MH 3G11 and MH 4H2. Averaged OD450 readings were plotted. Thechimeric anti-p53 R248Q antibodies were used at a concentration of 1ng/μl, while 3G11 and 4H2 hybridoma supernatants were tested at neatconcentration. Both MH 3G11 and MH 4H2 specifically recognized anddetected only human p53 R248Q full length protein over human p53 R175Hfull length, human p53 R273H full length, human p53 wild-type fulllength, and mouse p53 wild-type full length protein (single asterisks).Hybridoma supernatants and commercial anti-p53 antibody (1C12) wereincluded as positive control antibodies. (n=3).

FIG. 68 . Images showing detection of R273H mutant p53 in vivo by MH13E4. 100 ug of fluorescently-labeled R273H specific mAb MH 13E4 or IgGcontrol antibody, was injected i.v. in p53^(R273H) mutant HT29 tumorbearing mice. Mice were imaged by IVIS Spectrum in vivo imaging systemfor trafficking mAbs 72 hours following antibody injections.

FIG. 69 . Images showing detection of R175H mutant p53 in vivo by MH 4H5and MH 7B9. 100 ug of fluorescently-labeled R175H specific mAb, MH 4H5or MH 7B9 or IgG control was injected i.v. in p53^(R175H) clone 32 tumorbearing mice. Mice were imaged by IVIS Spectrum in vivo imaging systemfor trafficking mAbs at 6 h, 24 h, Day 2, Day 3 and Day 7 followingantibody injections. The bioluminescent light indicates the location ofthe clone32 tumors.

FIG. 70 . Images showing detection of spontaneously arising murine R172Hmutant p53 in vivo by MH 4H5. 100 ug of fluorescently-labeled R175Hspecific mAb MH 4H5 was injected i.v. into tumour bearing mutantp53^(R172H) mice. Mice were imaged by IVIS Spectrum in vivo imagingsystem on Days 2 and 3 following injection of the antibody.

FIGS. 71A and 71B. Schematic and images showing inhibition of HT29xenograft tumour growth by treatment with mAb 13E4. (71A) Schematicrepresentation of the experimental schedule. (71B) Images showing tumoursize as determined by measuring average photon intensity.

FIGS. 72A to 72D. Schematic, bar chart, images and graph showinginhibition of HT29 xenograft tumour growth by treatment with mAb 13E4.(72A) Schematic representation of the experimental schedule. (72B) Barchart showing tumour mass at the end of the experiment. (71C and 71D)Images and graph showing tumour size as determined by measuring averagephoton intensity.

FIGS. 73A and 73B. Schematic and bar chart showing therapeutic effectfor anti-p53 mutant R175 antibody against murine p53 R172H-positivecancer. (74A) Schematic representation of the experimental schedule.(74B) Bar chart showing tumour mass at the end of the experiment.

FIGS. 74A to 74F. Schematic, graphs, images and bar chart showingability of TrxR175H immunisation to raise antibodies capable of bindingto mutant p53 R175 protein. (74A) Schematic representation of theexperimental schedule. (74B) Graphs showing the ability of the serumfrom immunised mice to bind to the indicated antigen, as determined byELISA. (74C and 74D) Images showing ability of the serum from immunisedmice to bind to cells expressing p53 mutant R175H (74C), but notp53-negative TKO cells (74D). (74E) Images showing p53 mutantR175H-reactivity of serum obtained from mice immunised with TrxR175H.(74F) Bar chart showing the percentages of different immune cell subsetsin the splenocytes of mice immunised with the indicated antigens, asdetermined by flow cytometry.

EXAMPLES

The inventors describe in the following Examples the generation andcharacterisation of monoclonal antibodies against DBD region pointmutants of P53. The antibodies are shown to be highly specific for theindividual p53 hot-spot mutations, and the utility of the antibodies ina variety of biochemical and histological assays is demonstrated.

Example 1: Immunogen Design and Production

Attempts to generate antibodies against specific p53 mutants using alarge array of protocols have not been entirely successful, due likelyto lack of efficient expression of the mutant epitopes, resulting lackof specificity for the mutant p53 polypeptides.

The inventors therefore utilized the TrxA protein with a protrudingbody, in which was placed three copies of the mutant p53 mutation (i.e.R175H, R248Q or R273H), with variable lengths of the amino acid sequenceflanking the mutation. The mutant p53 polypeptide amino acid sequenceswere inserted into the active site of TrxA with flanking flexibleGly-Ser-Gly-Ser-Gly (SEQ ID NO:236) linkers separating the antigensequence and the TrxA sequence. Shorter Gly-Ser-Gly linkers were alsoinserted between each mutant p53 sequence.

TrxA is a widely-used fusion partner in Escherichia coli expressionsystems for enhancing protein expression levels, solubility and thermalstability, in which the active site (Cys33-Gly34-Pro35-Cys36) (SEQ IDNO:91) protrudes from the protein body into solution (LaVallie et al,2000 Methods Enzymol 326: 322-340; Young et al., 2012 Biotechnol. J.7:620-634). The presence of a restriction (RsrII) site on the DNAsequence coding for this active site provides an insertion point forinternal peptide fusions which can be presented on the surface of TrxA,and has been successfully exploited for production of antibodies byinsertion of the antigen within the solvent-accessible loop on the TrxAscaffold (Barrell et al., 2004 Protein Expr. Purif. 33:153-159).

TrxA scaffold harbouring the mutant p53 (R175H, R273H and R248Q)tri-peptide sequences cloned into the pJexpress404 vector, were obtainedfrom DNA2.0 (Menlo Park, Calif., USA). The coding sequences weredesigned with a C-terminus His6-tag to facilitate protein purificationby immobilized metal affinity chromatography (IMAC) and custom optimizedto E. coli preferred codons. Authenticity of the synthetic codingfragments was verified by DNA sequencing.

The amino acid sequences for the immunogens used to raise the antibodiesare shown in FIG. 1A to 1C.

Homology modeling predicted that inserting the mutant p53 antigensequences into the active site of the TrxA scaffold offers a viablepresentation strategy to increase the immunogenicity of the peptidesequence, with the mutant p53 antigen sequence extending away from theTrxA protein, and with the mutated residues exposed in the solventaccessible loop. The predicted 3D structure of the TrxR175H, TrxR248Qand TrxR273H immunogens by Swiss Model are shown (FIG. 2A to 2C),revealing the exposed, solvent accessible mutant p53 antigenic regions.

E. coli host strain BL21 (DE3) trxB (Novagen, Merck Millipore,Darmstadt, Germany) was used for the expression of the recombinantTrxp53 mutant constructs. Mini-scale expression studies to determinesolubility of the protein were performed as described in Liew et al.2014 Biochimie 89, 21-29. For large scale purification, recovery solubleand insoluble peptides were recovered by native and denaturing IMAC withintegrated on-column refolding into phosphate buffer respectively,performed as described in Liew et al. 2014. Protein quantitation wasperformed using the BCA assay (Pierce, Rockford, Ill., USA) and puritywas assessed by SDS-PAGE analysis.

Expression of the TrxR175H immunogen was induced in E. coli, and wasexpected to be of ˜18.8 kDa for TrxR175H, which was expressed in boththe soluble and insoluble forms (FIG. 3A), with higher partitioning intothe insoluble fraction. Analysis by mass spectrometry of purifiedTrxR175H revealed an actual molecular mass of 18,700 Da, consistent withloss of the first methionine residue (calculated mass 18,707 Da) (FIG.4A). Previous reports have shown that TrxA fusions containing a Serresidue in the second position of its amino acid sequence allowed forefficient cleavage of the first methionine (131 Da), presumably byendogenous methionine aminopeptidase in E. coli (Liew et al., 2007).Similarly, TrxR273H was expressed primarily in inclusion bodies in theinsoluble fraction, at the expected size of 18,200 Da (FIG. 3C). TheTrxR248Q was found to partition to the insoluble protein fraction, andmigrated at an unexpectedly lower molecular weight position of 15 kDa onSDS-PAGE (FIG. 3B), and mass spectrometric analyses revealed a mass of17,352 Da (FIG. 4B). The coding sequence of the construct was verifiedby DNA sequencing and the expressed protein could be purified byimmobilized metal affinity chromatography (IMAC), which indicated thatthe His6 tag located at the carboxyl terminus of TrxR248W was intact andthat the full length protein was translated, and suggested that thelower molecular mass may be a result of unexpected proteolytic cleavageby E. coli proteases, as has been demonstrated (Carrio et al., 1999Biochim. Biophys. Acta 1434, 170-176; Corchero et al., 1997 Biochem.Biophys. Res. Commun. 237, 325-330), downstream of Ala20 at theN-terminus and corresponds to a 166-residue protein moiety and itssodium form (17,324/17,347 Da). In any case, the unexpected proteolysisby E. coli proteases highlights a distinct advantage of nesting theantigen sequence within TrxA instead of the terminal regions of thefusion protein.

Example 2: Initial Screening to Identify p53 Monoclonal AntibodiesAgainst Specific p53 Mutants

Three groups of five, 8 week old Balb/c female mice (Biological ResourceCenter, Singapore) were inoculated with the Trxp53 mutant peptides. Thefirst immunisation was performed intraperitoneally with Sigma AdjuvantSystem (Sigma), followed by five intraperitoneal and subcutaneousinjections at 3 week intervals. One week after the fourth immunization,blood was taken from each mouse via cheek bleed using a lancet(MEDIpoint International Inc.). Blood samples were centrifuged for 10min at 1600 rpm and serum was aspirated and stored at 4° C., forsubsequent enzyme-linked immunosorbent assay (ELISA) analyses againstthe full length R175H, R273H and R248Q mutant p53 proteins.

The mouse with the highest serum antibody titer was selected as thespleen donor for fusion. The selected mice (one for each p53 mutation)received a final boost by intravenous injection of the Trxp53 mutantpeptide without adjuvant. Mouse myeloma SP2/0 cell line was used as thefusion partner. One week before fusion, cells were cultured in RPMI(Gibco) and 10% FBS until they attained >70% confluency in thelogarithmic phase. The spleen cells of the immune mice were removedunder sterile conditions. Generation, selection and cloning of hybridomacells were performed using the ClonaCell-HY Hybridoma Cloning kit(STEMCELL Technologies) according to the manufacturer's protocol.

Hybridoma clones secreting anti-mutant p53 mAbs were selected by ELISAwith 96-well plates coated with recombinant full length p53 proteinharboring the R175H, R273H and R248Q mutations respectively. Thioredoxinpeptide was used as negative control. Supernatant collected fromindividual hybridoma wells were tested on ELISA plates. 10% fecal bovineserum (FCS) was used for blocking and antibody dilution. PBS with 0.05%Tween 20 was used for washes. After washing, IgGs were detected using1:5000 goat anti-mouse IgG conjugated to HRP (Biorad) in PBST with 10%FCS. Plates were developed with 1×TMB ELISA substrate solution (Sigma).Absorbance was measured at 650 nm with EnVision Plate Reader (PerkinElmer).

Data from at least three independent mice are presented for eachmutation. Initial ELISA screening using the mutant or wild-type p53protein or the mutant p53 peptide fragment revealed that for each of thehybridomas producing antibody recognising mutant p53, the antibodieswere specific for the respective mutations and did not cross-react withthe wildtype p53 protein (FIG. 5A-5C). The antibodies were moresensitive to the whole mutant p53 protein as compared to the peptidefragments.

Further analyses using all peptides and mutant proteins to furtherconfirm specificity also showed that the three hybridoma clonesproducing monoclonal antibody against the R175H mutant p53 were highlyspecific (clones 7B9, 1008 and 4H5), and did not cross react with theR248Q or R273H mutant proteins and peptides (FIG. 6A). Similar resultswere obtained with the clones against the R248Q (clones 4H2 and 3G11)and R273H (clone 13E4) (FIGS. 6B and 6C).

To determine the epitopes targeted by the individual hybridoma clones,peptide phage display analysis was performed using the antibodiesagainst the three p53 mutants.

An M13 phage library (New England Biolabs) encoding random 12-merpeptides at the NH2 terminus of pill coat protein (2.7×109 sequences)was used. 50 nm purified antibody was coated on 96 well maxisorp plates(Nunc). The wells were incubated with blocking buffer (PBS, 0.5%Tween20, 2% BSA) for 1 h at room temperature, washed with washing buffer(PBS, 1% Tween20, 2% BSA), and incubated in washing buffer at roomtemperature with 4×10¹⁰ phages. Bound phages were eluted with 0.2 Mglycine (pH 2.2) and neutralized with 1 M Tris (pH 9.1). The elutedphages were amplified according to the manufacturer's instructions.

The selection process was repeated for three cycles. Phage plaques fromthe final round were selected, amplified as described by themanufacturer and sequenced. The peptides displayed on the selectedphages were deduced from analysis of results from DNA sequencing.Epitopes targeted by individual antibodies were obtained bydetermination of consensus sequences from alignment of peptide sequencesusing Clustal Omega multiple sequence alignment tool.

For the hybridomas against the R175H mutant p53, the consensus sequencewas “HCPHH”, in which the first Histidine was the mutation that replacesthe Arginine residue in the wildtype p53 (FIG. 7A). Almost all clonesfrom all hybridomas against R175H mutant p53 captured this sequence(FIG. 8A). The consensus sequences for the R248Q antibody clones were“SV . . . HY” (positions 215-216 and 233-234; FIG. 8A) for clone 3G11,and “RP” (positions 249-250; FIG. 7B) for clone 4H2. The consensussequence for anti-R273H clone 13E4 was “VH” (positions 272-273; FIG.7C).

To determine the crucial amino acids of the epitope, two sets ofindividually alanine substituted 13 amino acid peptides correspondingfrom Met169 to Arg181 (MTEVVRHCPHHER) of R175H mutant p53 protein, andArg267 to Gly 279 (RNSFEVHVCACP) of R273H mutant p53 protein werechemically synthesised and obtained from Bio Basic Inc. Peptides wereconjugated with an N-terminal Biotin and individually incubated on a 96well Streptavidin coated ELISA plate (Pierce, Thermo Scientific) at 10ug/ml for 1 hour. After three rounds of washing, the plates wereincubated with anti-R175H 10C8, 4H5 and 7B9 and anti-R273H 13E4respectively at 1 ug/ml for overnight at 4° C. The plates were incubatedfor an hour at 37° C. with secondary anti-mouse IgG-HRP after washing.After incubation, plates were washed three times prior to application ofsoluble HRP substrate for 5 minutes and absorbance at 650 nm wasdetermined with Envision plate reader (Perkin Elmer).

The results for the alanine scans for the R175H antibody clones 4H5, 7B9and 1008 are shown in FIGS. 9A-9C, and the results for the R273Hantibody clone 13E4 are shown in FIG. 9D. Mutations within p53 peptidesequences were identified as critical to the mAb epitope if they did notsupport reactivity of the test mAb but did support reactivity of otherantibodies.

BALB/c mice were given a single 0.25 mL intraperitoneal (IP) injectionof Incomplete Freund's Adjuvant (Sigma Chemical Co.). Fourteen dayslater, mice were injected with a single IP injection of 4×10⁵ in avolume of 0.5 mL of the hybridoma cells, after which they were examineddaily for development of ascites fluid as determined by abdominaldistention. Seven to ten days after the injection of hybridoma cells,mice were anesthetized and the ascites fluid was collected asepticallyfrom anesthetized mice by abdominal paracentesis with an 18-22 gaugeneedle by gravity flow into sterile centrifuge tubes. Digital pressurewas gently applied to the abdomen and the position of the mouse wasaltered as needed to facilitate removal of the ascites fluid. Asciteswas pooled for each individual cage of mice. The isotype of the antibodyclones was determined from hybridoma supernatant using a mouse mAbisotyping kit (Roche) according to the manufacturer's instructions. Theascitic fluids were diluted at a ratio of 1:10 with PBS and IgGs werepurified via Protein G column chromatography (GE Healthcare). Antibodywas eluted from the column through 5 ml of elution buffer containing0.2M Tris-Glycine pH2.7. The eluted fractions were dialyzed against 0.05mM PBS, pH 7.4. Confirmation of the purified antibodies was performed bySDS-PAGE under reducing conditions.

Example 3: Evaluation of Specificity of mAbs Against Specific p53Hot-Spot Mutants by Biochemical Approaches

The first attempt to determine specificity of the p53 hot-spotmutant-specific antibodies were made by evaluating their effectivenessin immunoblot assays. p53 null H1299-cells stably expressing ortransiently transfected with the six common hot-spot p53 mutations(R175H, R245S, R248Q and R248W, R249S, R273H and R282W) were used forthe initial analysis. 50 μg of cell lysates were loaded into each wellof a 4-12% Bis-Tris SDS polyacrylamide precast gels (Invitrogen). Theprotein marker used was Precision Plus Protein™ Standards Dual Colour(Bio-Rad, Hercules, Calif.). SDS-PAGE gels were ran at constant voltageof 60 volts (V) until the protein bands exceeded the stacking gel, afterwhich the gel was continuously ran at 100V until the dye front reachesthe bottom. For immunoblotting, protein transfer was carried out on theiBlot™ Drying Blotting system (Invitrogen) for 10 minutes at 20-25V ontonitrocellulose membranes. The membrane was washed three times for 10minutes each with PBST (phosphate buffered saline (PBS) containing 0.05%Tween20 (Bio-Rad, Hercules, Calif.), and non-specific binding wasblocked using 4% non-fat milk in PBST buffer for 1 hour with gentleagitation. Subsequently, the membrane was washed three times for 10minutes each with PBST. Excess PBST after the washing step was removedbefore hybridoma supernatant was added. The primary antibody wasincubated under gentle agitation at 4° C. overnight. The membrane waswashed three times for 10 minutes each with PBST to remove unboundprimary antibodies. 1:5000 goat anti-mouse IgG conjugated to HRP(Biorad) in PBST with 10% FCS was used for detection. The secondaryantibody was incubated under gentle agitation for 1 hr at roomtemperature. Unbound secondary antibodies were washed off in the abovementioned manner before visualization using Clarity western blot ECLsubstrate (Biorad). Densitometric analysis was performed using OdesseyFc (Licor).

All three mutant-specific antibodies were able to detect theircorresponding mutant p53 proteins expressed in H1299 cells, withoutdetecting the other mutants or the wildtype p53 that were abundantlyexpressed, as determined using the pan-p53 antibody DO1 (FIG. 11 ).

Of particular significance was the finding that the anti-R248Q antibodywas unable to detect the closely related R248W mutant, which comprises adifferent mutation at the same residue, highlighting the very highspecificity of the antibodies.

Immunoprecipitation analyses were also performed using the p53mutant-specific antibodies, followed by detection with the anti-p53rabbit antibody CM1, which again revealed that the antibodies werespecific in bringing down only the respective mutant proteins (FIG. 12). Reverse immunoprecipitation with CM1 antibody followed byimmunoblotting with the mutant-specific antibodies also gave similarresults, with extreme specificity (FIG. 12 ).

The inventors next determined the ability of these mAbs to recognizeendogenous p53 in a large number of human tumor cell lines that expressthe wild-type protein or the various hot-spot mutants (FIG. 13 ). Directimmunoblotting with the mutant specific mAbs was able to detect only therespective mutant p53 proteins in the tumor cell lines, though all celllines expressed large amounts the various mutant p53, or the wild-typep53 that was induced by UV-irradiation and detected by the DO1 antibody(FIG. 14 ), confirming the specificity of the antibodies.

Immunoprecipitation of the endogenous proteins indicated the same trendfor the R175H-specific antibody (FIG. 15 , left panel). Both the R248Qand the R273H antibodies were also able to specifically detect onlytheir respective mutant proteins when used for immunoblot detectionafter the primary immunoprecipitation with the pan-p53 CM1 antibody(FIG. 15 , middle and right panels). However, when used directly forimmunoprecipitation followed by immunodetection with CM1, these twoantibodies detected some other p53 mutants as well, likely due tonon-specific binding under the conditions tested. Nonetheless, the highlevel of specificity in direct immunoblotting and uponimmunoprecipitation with a pan-p53 antibody highlights the specificityof these antibodies against their respective mutant p53 antigens.

Example 4: Specificity of mAbs Against Specific p53 Hot-Spot Mutants inImmunofluorescence Analyses

To test the specificity of the mutant-specific antibodies byimmunofluorescence staining, the inventors again utilized theH1299-cells overexpressing the wild-type p53 or the various p53 mutants,or tumor cells lines that express endogenous mutant p53 proteins.

Fixed, transfected cells on 96 well plates were subjected topermeabilization with 0.4% Triton X-100 for 20 minutes. After rinsingwith PBS, cells were blocked with 5% BSA in PBSTritonX (PBSTX) for 20min, followed by incubation in hybridoma supernatant at 4° C. overnight.IgGs were detected using 1:1000 goat Alexaflor 488 Donkey anti-mouse IgGconjugated (Life technologies) in PBSTX with 1% BSA. Then, cells werecounterstained with DAPI, and viewed with Incell Analyzer (GEHealthcare).

A distinct nuclear staining pattern was observed with themutation-specific antibodies, which detected only their respectivemutant proteins when overexpressed (FIG. 16 ), or in the endogenousstate in tumor cell lines (FIGS. 17A and 17B), though all cellsexpressed the various p53 forms in abundance, as determined by stainingwith either the DO1 or the CM1 antibodies.

As observed with analysis by immunoblot and immunoprecipitation, theantibody against the R248Q mutant was extremely specific, and was unableto detect the related R248W mutant protein (FIG. 17B).

Example 5: Analyses of Human Tumor Samples Using mAbs Against theSpecific p53 Hot-Spot Mutants by Immunohistochemistry

To determine the effectiveness of the p53 mutant-specific antibodies inparaffin-embedded tissues, the inventors analyzed a large number ofhuman tumor samples by immunohistochemical (IHC) analysis. Firstly, aseries of tumor samples with known p53 mutations, confirmed by DNAsequencing (FIG. 18A), were examined using the mutant-specificantibodies.

Mouse tumour sections from HT29 xenograft mouse model with p53 R273Hgenotype and tumour sections generated from p53R172H mutant cell lineswere processed into paraffin blocks by the Advanced Molecular PathologyLaboratory (AMPL), Institute of Molecular and Cell Biology. Wax sectionsof 5 μm were then embedded onto glass slides (Leica Biosystems) anddried for an hour on a 50° C. hot plate. Sections were deparaffinized inxylene (ChemTech Trading) and rehydrated through descending percentagesof ethanol (ChemTech Trading) into water. Tissue sections were heatedwith Target Retrieval Solution, pH9 (Dako) for antigen exposure, thenrinsed in PBS. Endogenous peroxidase was blocked with 2% (v/v) hydrogenperoxide (Merck) in PBS for 30 min, rinsed with PBS. Sections wereblocked with 10% (v/v) goat serum (Dako) in PBS for 1 h then incubatedwith biotinylated primary antibodies clones at 4° C. overnight. Sectionswere washed with water then rinsed in PBS before detection withstreptavidin-HRP antibody (Bio Legend). Antigen-antibody interaction wasthen visualized using 3,3-diaminobenzidine as a substrate, and thesections were lightly counterstained with hematoxylin before dehydratingand mounting in Cytoseal 60 synthetic resin (Richard-Allan Scientific™,Fisher Scientific). Slides were imaged under bright field using theAxioImager (Zeiss) light microscope and analyzed with AxioVision Rel 4.8software (Carl Zeiss AG).

As was the case in the immunofluorescence analyses, the threemutant-specific antibodies stained the samples with the respectivemutations in p53, but not the sample with either wild-type p53 or withother mutations (FIG. 18B), demonstrating specificity in the IHCsetting.

The inventors further evaluated several tumor microarrays from colon,breast (triple negative), lung, prostate and renal tumors by stainingwith these antibodies. Representative results from staining with thepan-p53 antibody suitable for IHC staining (DO7) and the R175H-specificantibodies are shown (FIG. 19 ). Three groups of samples emerged: onethat was stained both by DO7 and the R175H mAb; one that was onlypositive for DO7; and the last that was negative for both antibodies(FIG. 21 ). The highest level of DO7 staining was for in the triplenegative breast, lung and colon cancer groups, confirming previous datathat these cancers have a higher mutation rate for p53 (Olivier et al.,2004 IARC Sci Publ 157:247-270), and are therefore positive for stainingby anti-p53 antibodies. Staining by the R175H-mAb mirrored that of DO7in the first group, suggesting that both the antibodies were recognizingthe same cells in this group. A few samples were sequenced to determinethe p53 mutational status, and found that all samples that were stainedby both antibodies carried a R175H mutation. By contrast, samples thatwere stained by DO7 only had mutations in other residues of p53 but noton R175, and the samples that were negative for both antibodies had nomutations in p53.

Human triple negative breast cancer samples B41, B89, B98, B27 and B52were stained with anti-p53 antibodies DO7, 11 D1, or the anti-R175Hantibody for analysis by immunohistochemistry. Tumor samples B41, B89and B98 comprising the R175H mutation (CAG—underlined) were positive forstaining by all three antibodies (FIG. 20A). Tumor sample B27 notcomprising the R175H mutation (CGC—underlined) was only stained by theanti-p53 antibodies DO7 and 11 D1 (and not by the anti-R175H antibody),and sample B52 was not stained by any of the anti-p53 antibodies (FIG.20B).

These data collectively indicate the specificity of thesemutant-specific antibodies in paraffin-embedded clinical samples.

Example 6: Comparison of Effectiveness of the Human p53 Mutant-SpecificAntibodies with Equivalent Mouse Mutants

Finally, the inventors investigated if the antibodies are able to detectthe corresponding mouse mutations. The human sequences corresponding tothe three mutations studied here are highly homologous in mouse p53(FIG. 22 ), and the sequences around the R175 residue are similar to theequivalent mouse R172 residue (Olive et al, 2004 Cell 119, 847-860).Hence, the inventors utilized mouse embryonic fibroblasts (MEFs) fromthe R172H mice, which contain an equivalent mutation to the R175H inhumans (Lang et al., 2004 Cell 119, 861-872), or MEFs with an unrelatedmutation R246S, which is equivalent to the human R4249S (Lee et al.,2012 Cancer Cell 22, 751-764).

Direct immunoblotting indicated that the anti-R175H antibody was able todetect only the mutant p53 from the R172H MEFs, but not from the R246Sor the wild-type MEFs, although these latter cells expressed significantamounts of p53 as determined by the pan-p53 antibody, CM5 (FIG. 23A).Immunoprecipitation with the R175H-specific antibody followed byimmunoblotting also demonstrated that the antibody was indeed specificand can detect the mouse R172H mutant protein (FIG. 23B). The inventorsalso tested the ability of the R175H-specific antibody byimmunofluorescence (FIG. 23C) and IHC (FIG. 23D) analyses, whichconfirmed its specificity, highlighting the utility of these antibodiesin different species.

Immunostaining for IHC analyses was performed on formalin fixedparaffin-embedded (FFPE) 5 μm sections. Antigen retrieval was performedusing with Dako Tris/EDTA target retrieval solution, pH9. Blocking wasperformed with DAKO 10% goat serum. Secondary antibody was DakoEnvision™+/HRP. Develop with DAKO liquid DAB+. Images were captured witha Zeiss AxioImager upright microscope using 40× objective lens.

Example 7: Monoclonal Anti-Mutant p53 Antibodies

7.1 R175H Mutant p53 Antibody Clones 4H5, 7B9 and 10C8

Anti-R175H p53 mouse monoclonal antibodies were raised by immunisingmice with immunogen comprising three copies of the R175H p53 mutationinserted in the active site sequence of TrxA, as described in Example 1(see FIG. 1A).

Hybridoma clones producing anti-R175H p53 mouse monoclonal antibodieswere obtained. The amino acid sequences of the light and heavy chainvariable domain sequences were determined and are shown in FIGS. 24 and25 . The DNA sequences encoding the light and heavy chain variabledomain sequences for the antibodies are shown in FIG. 27 .

The CDRs were predicted using VBASE2 (Retter et al. Nucleic AcidsResearch (2005) 33 (Database issue):D671-674, incorporated by referencehereinabove).

7.2 R248Q Mutant p53 Antibody Clones 3G11 and 4H2

Anti-R248Q p53 mouse monoclonal antibodies were raised by immunisingmice with immunogen comprising three copies of the R248Q p53 mutationinserted in the active site sequence of TrxA, as described in Example 1(see FIG. 1B).

Hybridoma clones producing anti-R248Q p53 mouse monoclonal antibodieswere obtained. The amino acid sequences of the light and heavy chainvariable domain sequences were determined and are shown in FIGS. 28 and29 . The DNA sequences encoding the light and heavy chain variabledomain sequences for the antibodies are shown in FIG. 31 .

The CDRs were predicted using VBASE2 as above.

7.3 R273H Mutant p53 Antibody Clone 13E4

Anti-R273H p53 mouse monoclonal antibodies were raised by immunisingmice with immunogen comprising three copies of the R273H p53 mutationinserted in the active site sequence of TrxA, as described in Example 1(see FIG. 10 ).

A hybridoma clone producing anti-R273H p53 mouse monoclonal antibodieswas obtained. The amino acid sequences of the light and heavy chainvariable domain sequences were determined and are shown in FIGS. 32 and33 . The DNA sequences encoding the light and heavy chain variabledomain sequences for antibody clone 13E4 are shown in FIG. 34 .

The CDRs were predicted using VBASE2 as above.

Example 8: Characterisation of Monoclonal Anti-Mutant p53 Antibodies

8.1 R175H Mutant p53 Antibody Clones 4H5, 7B9 and 10C8

Western blot analysis was performed on cell extracts obtained from R172Hmouse thymic lymphoma cell line cells, T47D cells, WiDr cells and DKOcells using cell culture supernatant of hybridoma clones 4H5, 7B9 and1008. The results are shown in FIG. 35 . Antibodies from each clone werespecific for R175H mutant p53.

Immunofluorescence analyses were also performed using R172H mouse thymiclymphoma cell line cells, TKO cells, C6 cells, H1299 cells, T47D cells,and H1299 cells transfected with a construct expressing R175H mutantp53, using cell culture supernatant of hybridoma clones 4H5, 7B9 and1008. The results are shown in FIG. 36A to 36C. Antibodies from eachclone were specific for mouse R172H mutant p53.

The epitope recognised by the antibodies 4H5, 7B9 and 1008 is shown inthe context of human R175H p53 in FIG. 10 .

The antibody clones were further investigated for their ability torecognise R175H p53 by immunohistochemical (IHC) analysis of mouseintestinal tissue sections obtained from p53 knockout mice orirradiated, R172H p53 positive mice, using cell culture supernatant ofhybridoma clones 4H5, 7B9 and 1008. The results are shown in FIG. 37 .The antibodies were able to detect R172H mutant p53 in mouse intestinaltissue sections by IHC.

Antibody clone 4H5 was further analysed for ability to visualise andmonitor R175H-positive cancer in vivo. Mouse tumour cells derived fromP53^(R172H/R172H) mice were transfected with luciferase gene and used toestablish a tumour model. Mice were injected IV with 100 μg of XenoLightCF750-labelled anti-R175H mutant p53 antibody clone 4H5, and imaged byIVIS analysis at 6 h, 24, 72 h and 7 days post-injection. The results ofthe experiment are shown in FIG. 38 . The antibody was demonstrated tobe suitable for visualisation and monitoring tumour in vivo.

8.2 R248Q Mutant p53 Antibody Clones 3G11 and 4H2

Western blot analysis was performed on cell extracts obtained from TKOcells, and HCC70 cells (which possess the R248Q mutation) using cellculture supernatant of hybridoma clones 3G11 and 4H2. The results areshown in FIG. 39 . The antibodies were specific for R248Q mutant p53.

Immunofluorescence analyses were also performed using TKO cells, HCC70cells and OVCAR3 cells (which possess the R248Q mutation), using cellculture supernatant of hybridoma clones 3G11 and 4H2. The results areshown in FIGS. 40A and 40B. Antibodies from each clone were specific forR248Q mutant p53.

The epitopes recognised by antibody clones 3G11 and 4H2 were analysed bypeptide phage display analysis (FIGS. 8B and 7B). The epitopesrecognised by the antibodies are shown in the context of human R248Q p53in FIG. 10 .

8.3 R273H Mutant p53 Antibody Clone 13E4

Western blot analysis was performed on cell extracts obtained from T47Dcells and MB468 cells (which possess the R273H mutation) using cellculture supernatant of hybridoma clone 13E4. The results are shown inFIG. 41 . Antibody from clone 13E4 was specific for R273H mutant p53.

Immunofluorescence analysis was also performed using T47D cells, andWiDr cells expressing R273H mutant p53, using cell culture supernatantof hybridoma clone 13E4. The results are shown in FIG. 42 . Antibodyfrom clone 13E4 was specific for R273H mutant p53.

The epitope recognised by 13E4 antibody is shown in the context of humanR273H p53 in FIG. 10 .

Antibody clone 13E4 was further analysed for ability to visualise andmonitor R273H-positive cancer in vivo. Cells of the p53-R273H-mutantHT29 tumour cell line were transfected with luciferase gene and used toestablish a tumour model. Mice were injected IV with 100 μg of XenoLightCF750-labelled anti-R273H mutant p53 antibody clone 13E4, and imaged byIVIS analysis at 72 hours post-injection. The results of the experimentare shown in FIG. 43 . The antibody was demonstrated to be suitable forvisualisation and monitoring tumour in vivo.

Example 9: Chimeric Mouse Fv-Human IgG1 Fc Anti-Mutant p53 Antibodies

Mouse Fv-Human IgG1 Fc chimeric versions of the anti-mutant p53antibodies were prepared.

Variable regions of the heavy and light chains were cloned from parental4H5, 7B9, 1008, 3G11, 4H2 and 13E4 mouse monoclonal antibody clones intopTT5 vectors each containing the human IgG1 constant region.

Mouse-human chimeric heavy- and light-chain plasmids were co-transfectedinto HEK293-6e cells at 1 μg total plasmid per million cells, using 2 μLof 293-fectin transfection reagent per μg of plasmid. Culturesupernatant containing secreted chimeric antibodies was harvested andpurified using protein G agarose beads, 4 to 6 days post-transfection.

Chimeric antibody was eluted off beads using 0.1 M glycine-HCl (pH 2.7)neutralised with 1 M Tris (pH 9.0) and dialysed into PBS.

The chimeric antibodies were determined to be able to recognise theirrespective mutant p53 by ELISA (test concentration 1 ng/μL).

Mouse-Fv-Human IgG1 Fc chimeric versions of anti-R175H p53 antibodyclones 4H5, 7B9 and 10C8 were also investigated for ability to recogniseR175H p53 by IHC analysis of mouse intestinal tissue sections obtainedfrom irradiated, R172H p53 positive mice. The results are shown in FIG.44 . Chimeric versions of the antibodies were able to detect R172Hmutant p53 in mouse intestinal tissue sections by IHC.

Example 10: Analysis of the Ability of Anti-p53 Mutant Antibody to TreatCancer In Vivo

The inventors investigated the ability of the monoclonal anti-mutant p53antibodies to treat cancer in vivo.

Briefly, mice were injected with 5×10⁶ SKBR3 cells (human breast cancercells) carrying a luciferase gene. SKBR3 cells carry the R175H mutationin p53 (see e.g. FIG. 13 ). The luciferase gene allowed monitoring oftumor growth in vivo, by detection of luciferase activity. Four daysafter mice have been injected with the SKBR-3 cells, mice were treatedeither with 100 μl of anti-R175H antibody, or 100 μl of IgG antibodycontrol. Injections were repeated every four days (as indicated by thearrows in FIG. 45 ). Tumor growth was monitored throughout theexperiment by measuring luciferase luminescence. The results are shownin FIG. 45 , which demonstrates an anti-cancer effect in mice treatedwith the anti-R175H antibody, as evidenced by reduced levels ofluciferase activity as compared to the control treatment group.

Example 11: Analysis of the Ability of the Immunogens to be Used toGenerate an Immune Response to Mutant p53 Polypeptides

The inventors also investigated the ability of the immunogens describedin Example 1 to be used as vaccines, for stimulating an immune responseagainst p53 mutant polypeptides.

Groups of mice were immunised with the immunogens, and the polyclonalantibody response was analysed by ELISA to determine whether theimmunogens can be used as vaccines to trigger antibody responses.

The results are shown in FIGS. 46A-46E. Immunization with the differentmutant p53 immunogens is shown to induce the production of antibodieswhich are highly specific for peptide corresponding to the respectivep53 mutant (FIGS. 46A, 46C and 46E), and which display minimalinteraction with wildtype human p53 (FIGS. 46B, 46D and 46F). Theseresults demonstrate that the mutant p53 immunogens are capable oftriggering an antibody response, and that this immunity is specific tothe respective p53 mutant.

The inventors next investigated whether the antibodies generated inresponse to immunisation with the immunogens were capable of recognisingmutant p53 polypeptides. Sera was obtained from mice injected with theR175H, R248Q or R273H immunogens described in Example 1, and analysedfor ability to recognise mutant p53 polypeptides by immunofluorescenceanalysis. The results are shown in FIGS. 47A-47C, which demonstrate thatthe antibodies generated in mice in response to immunisation withimmunogen were capable of recognising the corresponding mutant p53polypeptide.

Example 12: Conclusion

The inventors have successfully generated p53 mutant-specific antibodiesagainst three commonly-occurring p53 hot-spot mutations in the DBDregion—the R175H, R248Q and R273H (Vikhanskaya et al., Nucleic Acids Res(2007) 35:2093-2104). The antibodies are characterised, and theirutility in a variety of biochemical and histological assays isdemonstrated, as is their usefulness to treat cancer in vivo.

The inventors have for the first time been able to generate antibodiescapable of specifically binding to single point mutants of p53, which donot cross-react with wildtype p53, raised using immunogen in whichantigen expression is enhanced by the provision of multiple copies ofthe region containing the mutation, displayed by protrusion from theprotein body into solution, using TrxA as a fusion partner. Thisapproach consistently led to the generation of mAb clones againstseveral p53 mutants, with high specificity and selectivity.

The present experimental examples demonstrate generation of antibodiesto three of the most common hot-spot mutations found in p53, namelyR175H, R248Q and R273H. The mAbs generated against these mutants werespecific in their ability to discern between the intended antigens andother mutations or the wildtype p53 protein, in a variety of techniques,ranging from immunoblotting, immunoprecipitation, immunofluorescence andimmunohistochemistry. The inventors have moreover demonstrated theability to inhibit growth of tumor cells comprising the correspondingp53 mutation in a human xenograft cancer mouse model in vivo.

Furthermore, the antibodies were able to detect the correspondingmutations in other species such as mouse, as demonstrated with the R175Hmutation, and thus, should be applicable to the other mouse mutants,given the sequence conservation across species (see e.g. FIG. 22 ),thereby proving to be valuables tools for fundamental research.

The mutant-specific antibodies are useful tools to dissect out theindividual and combined roles of both the wild-type and mutant p53proteins in the same cell, potentially even at the single cell level,and during the clonal evolution of the cancer cell.

The utility of the mutant-specific mAbs for IHC analysis of human tumorsamples highlights that these mAbs are very useful tools in pathologicalanalyses in determining p53 status, which could be easily implementedand is significantly cost effective compared to DNA-sequencingtechnologies.

The TrxA presentation system utilised in the present examples will beuseful for generating mAbs against other mutations in p53 which can beof clinical utility, and also for generating mAbs against othermutations found in tumor suppressors and oncogenes. Monoclonalantibodies which are able to discriminate between proteins differing byonly a single amino-acid could be clinically useful as diagnostic andtherapeutic agents.

Moreover, the inventors have demonstrated the usefulness of theimmunogens for vaccination strategy, demonstrating the ability to inducean antibody response capable recognising mutants of p53.

In a therapeutic context, such mutant-specific antibodies are likely tobe very safe as they will not have any side-effects in normal cells ofpatients that do not carry the mutation. It is expected that suchantibodies would be superior to the currently available generalantibodies against proteins that are either overexpressed or deregulatedin disease.

Example 13: Effect of Mutant-Specific Anti-p53 Antibody on Tumour GrowthIn Vivo

The inventors next investigated the effect of administration of amonoclonal p53 mutant-specific antibody on growth of cancer cells in invivo.

Briefly, SCID mice were injected subcutaneously at their flanks with5×10⁶ luciferase labelled SKBR cells (which carry the R175H mutation inp53—see e.g. FIG. 13 )). From day 4, mice were injected intravenouslyevery 4 days with 100 μl of monoclonal antibody specific for the R175Hmutant p53 (a175) or isotype control antibody (IgG). Tumour growth wasmonitored by measuring luciferase activity.

The results of the experiment are shown in FIG. 48 . A stronganti-cancer effect was observed in mice treated with the monoclonalantibody specific for the R175H mutant p53.

Example 14: Preparation of Mouse-Human Chimeric Anti-p53 MutantAntibodies

The inventors prepared chimeric mouse-human versions of the anti-R175Hantibody clones 4H5, 7B9 and 1008, the anti-R273H antibody clone 13E4and the anti-R248Q antibody clones 3G11 and 4H2.

DNA encoding the variable heavy and light chains of the parental mousemonoclonal antibodies were cloned from parental mouse monoclonalantibodies into separate pTT5 vectors each containing the human constantregion. Mouse-human chimeric heavy- and light-chain plasmids wereco-transfected into HEK293-6e cells at 1 μg total plasmid per 1×10⁶cells, using 2 μl of 293-fectin transfection reagent per microgram ofplasmid.

4-6 days after transfection, the cell culture supernatant containing thesecreted mouse-human chimeric antibodies was harvested, and theantibodies were purified using protein G agarose beads. The chimericmouse-human antibodies were eluted off beads using 0.1 M glycine-HCl (pH2.7) neutralised with 1 M Tris (pH 9.0) and dialysed into PBS. Themouse-human chimeric antibodies comprise mice Fv and human Fc.

Mouse-human chimeric anti-p53 mutant R175H antibody clone VL sequencesare shown in FIGS. 49 and 51 , and the VH sequences are shown in FIGS.50 and 52 .

Mouse-human chimeric anti-p53 mutant R273H antibody clone 13E4 VLsequence is shown in FIGS. 53 and 55 , and the VH sequence is shown inFIGS. 54 and 56 . Mouse-human chimeric anti-p53 mutant R248Q antibodyclone VL sequences are shown in FIGS. 57 and 59 , and the VH sequencesare shown in FIGS. 58 and 60 .

Example 15: Characterisation of Mouse-Human Chimeric Anti-p53 MutantAntibodies

Mouse-Human Chimeric Anti-p53 R175H Antibodies

The mouse human chimeric anti-p53 R175H antibodies were analysed byELISA for binding to R175H mutant p53.

The results are shown in FIG. 61 . The chimeric anti-p53 R175Hantibodies (MH 4H5, MH 7B9, MH 10C8) antibodies produced positivesignals between 0.1 and 1 ng/μl concentrations, bound specifically onlyto human p53 R175H full length protein and not human p53 R273H fulllength, human p53 wild-type full length, or mouse p53 wild-type fulllength protein. Commercial anti-p53 antibody (1012) was included as apositive control antibody.

Binding was also analysed by western blot, and the results are shown inFIG. 62 . Detection of denatured endogenous p53 in human cell lines viawestern blot (antibodies were used at a concentration of 1 ng/μl)confirmed the specificity of the chimeric anti-p53 R175H antibodies (MH4H5, MH 7B9, MH 10C8). The antibodies only detected p53 from the SKBR3cell line which harbours the R175H mutation in p53, and not from thep53-null H1299 cell line, the wildtype 53 cell lines MCF7 and A549, northe R273H cell lines A431 and SW480.

The antibodies were further analysed for their ability to bindspecifically to R175H mutant p53 by immunohistochemical analysis ofbinding to different cancer cell lines fixed in 4% paraformaldehyde andembedded in paraffin. The mouse human chimeric anti-p175H antibodieswere shown only to stain SKBR3 cells. Representative images from theanalysis using MH 7B9 are shown in FIG. 63 .

Mouse-Human Chimeric Anti-p53 R273H Antibody

The mouse human chimeric anti-p53 R273H antibody 13E4 was analysed byELISA for binding to R273H mutant p53.

The results are shown in FIG. 64 . The chimeric anti-p53 R175H antibodyMH 13E4 bound to human p53 R273H full length protein much more than itbound to thioredoxin, human p53 R175H full length and human p53wild-type full length protein.

Binding was also analysed by western blot, and the results are shown inFIG. 65 . Detection of denatured endogenous p53 in human cell lines viawestern blot (antibodies were used at a concentration of 1 ng/μl)confirmed the specificity of MH 13E4, which only detected p53 from theA431 and SW480 cell lines which harbour the R273H mutation in p53, andnot from the p53-null H1299 cell line, the wildtype 53 cell lines MCF7and A549, nor the R175H cell line SKBR3.

The antibody was further analysed for ability to bind specifically toR273H mutant p53 by immunohistochemical analysis of binding to differentcancer cell lines fixed in 4% paraformaldehyde and embedded in paraffin.MH 13E4 was found only to stain cells harbouring the R273H mutation(i.e. A431 cells)—see FIG. 66 .

Mouse-Human Chimeric Anti-p53 R248Q Antibodies

The mouse human chimeric anti-p53 R248Q antibodies were analysed byELISA for binding to R248Q mutant p53. The antibodies were used in theexperiments at at final concentration of 1 ng/μl.

The results are shown in FIG. 67 . The chimeric anti-p53 R248Qantibodies (MH 3G11 and MH 4H2) antibodies were found to bind tofull-length R248Q p53, and not to full-length R175H or R273H p53, orfull-length wildtype human or mouse p53. Commercial anti-p53 antibody(1C12) was included as a positive control antibody.

Example 16: Evaluation of Anti-p53 Mutant Antibodies as DiagnosticAntibodies for Tumor Imaging In Vivo

The inventors next investigated whether anti-p53 mutant antibodies wereuseful for tumor imaging in vivo.

Briefly, 100 ug of fluorescently-labeled R273H specific mAb MH 13E4 (orfluorescently labelled IgG control), was injected intravenously intomice bearing HT29 tumors (which harbour the R273H mutation in p53). Micewere imaged using the IVIS Spectrum in vivo imaging system fortrafficking mAbs 72 hours after antibody injection.

The results are shown in FIG. 68 . MH 13E4 specifically detected R273Hmutant p53-positive HT29 xenograft tumours in nude mice.

In a separate experiment, 100 ug of fluorescently-labeled R175H specificmAb MH 4H5 or MH 7B9, was injected i.v. into mice bearing R175H mutantp53-positive clone32 tumors. Mice were imaged using the IVIS Spectrum invivo imaging system for trafficking mAbs at 6 h and 24 h, and on Days 2,3 and 7 following antibody injection. The clone 32 cells expressluciferase, and so the location of the tumor cells could be analysed bydetection of luciferase activity.

The results are shown in FIG. 69 . MH 4H5 and MH 7B9 antibodies detectedR175H mutant p53 and were retained in the tumours for up to 7 days.Specificity of the antibodies for the tumour cells is demonstrated bydetection of luciferase at the same position as the antibodies.

In a further experiment, it was investigated whether anti-p53 mutantR175H antibodies could detect spontaneously arising R175H p53 tumours.100 ug of fluorescently-labeled R175H specific mAb MH 4H5 was injectedi.v. into mice having the murine R172H mutation in murine p53. Mutantp53^(R172H) mice are highly susceptible to the spontaneous developmentof tumours harbouring the R172H mutation in murine p53. Mice were imagedusing the IVIS Spectrum in vivo imaging system for trafficking mAbs onDay2 and Day3 following antibody injections.

The results are shown in FIG. 70 . MH 4H5 was able to detectspontaneously occurring mutant murine p53 R172H tumours.

Example 17: Evaluation of Therapeutic Utility of Anti-p53 MutantAntibodies to Treat Cancer In Vivo

The inventors next investigated whether the anti-p53 mutant antibodieswere useful as a treatment for cancer in vivo.

Anti-p53 Mutant R175H Antibodies

In a first experiment, the therapeutic effect of administration of themonoclonal antibody 13E4 was analysed in a HT 29-luciferase xenografttumor model. Briefly, nude Balb/c mice (n=3) were subcutaneouslyinoculated with 5×10⁶ HT29-luc cells on Day 0, and followed by i.v.injection of 15 mg/kg of control IgG or 13E4 mAb on Days 3, 7, 11, 14,18 and 21. Mice were analysed at Day 28. Tumour growth was determined bymeasuring average photon intensity.

A schematic representation of the treatment schedule is shown in FIG.71A. The results of the experiment are shown in FIG. 71B. Mice treatedwith 13E4 mAb showed an inhibition of 81.62% tumour size as compared tomice treated with IgG control antibody.

In a second experiment, the therapeutic effect of administration of themonoclonal antibody 13E4 was analysed in a HT 29-luciferase xenografttumor model. Briefly, nude Balb/c mice (n=3) were subcutaneouslyinoculated with 5×10⁶ HT29-luc cells on Day 0 on each flank, andfollowed by i.v. injection of 10 mg/kg of control IgG or 13E4 mAb onDays 4, 7, 11, 14, 18, 21, 25, 28, 32 and 35. Mice were analysed on Days7, 14, 21, 28 and 35. The tumour volume was measured every weekfollowing inoculation by luminescence imaging of luciferase expressingHT29 tumour cells. At the end of the experiments tumours were excisedfrom mice and the mass of the tumours was recorded.

A schematic representation of the treatment schedule is shown in FIG.72A, and the results of the experiment are shown in FIGS. 72B, 72C and72D. Mice treated with 13E4 mAb showed an inhibition of 87.3% tumoursize as compared to mice treated with IgG control antibody, and thetumor mass was approximately 2.5 time less than the weight of tumoursobtained from mice treated with control IgG. FIG. 72D shows inhibitionof tumor growth by 13E4 over time.

In a further experiment, the anti-p53 mutant R175H antibody 4H5 wasanalysed for its ability to inhibit growth of spontaneously-occurringmurine p53 mutant R172H-positive cancer. Briefly, a mouse tumor cellline, clone32, was generated from p53^(R172H) mutant mouse. 3×10⁶clone32 cells were injected into syngeneic B6 mice on Day 0, and 100 ugof 4H5, 7B9 (mAb specific to R175H), 13E4 (specific to R273H) and 11D10(reactive to both human and mouse p53) were i.v. injected into mice onDays 3, 6, 9, 12, 15, 18, 21 and 24. Mice were sacrifice on Day 25 foranalysis and tumor measurement.

A schematic representation of the treatment schedule is shown in FIG.73A, and the results of the experiment are shown in FIG. 73B. Theaverage of tumor weight in 4H5 (R175H mAb) treated mice was 75% lessthan the weight of tumors in the control IgG treated group. 7B9 (R175HmAb), 13E4 (R273H mAb), 11 D10 (p53 mAb) did not show significanteffects on the weight on the syngeneic mouse tumor.

Example 18: Evaluation of Immunogens Used to Raise p53 Mutant Antibodiesas Vaccine Candidates

The inventors next investigated whether the immunogens used to raise thep53 mutant-specific antibodies of the present invention were capable ofvaccinating subjects against the development of p53 mutant cancers.

Wildtype BALB/C or B6 mice and mutant p53^(R172H) mice were injectedwith TrxR175H protein (see Example 1) on Days 0, 21, 42, 63 and 84.Serum was collected 7 days after each injection and analysed by ELISA,cell staining and western blot. Antigens used for ELISA analysis werethioredoxin protein (Trx), Trx-R175H protein or the full-length R175Hmutant p53 protein. On day 87, p53 R175H-positive tumor cells wereinjected into the mice.

A schematic representation of the experimental procedures is shown inFIG. 74A. The results of the ELISA analysis are shown in FIG. 74B, andshow that serum obtained at the indicated time points (after the firstand second immunizations) reacted with TrxR175H protein and thefull-length R175H mutant p53 protein.

FIGS. 74C and 74D show the results of staining of SKBR3 cells (whichharbour the R175H mutation) and TKO cells (p53 null) with serum obtainedfrom the first and second bleeds from 10 different mice (M #1-M #10).Antibody in the serum of TrxR175H immunized mice showed positivestaining on p53^(R175H) expressing SKBR3 cells but not the p53 knock outTKO cells.

FIG. 74E shows the results of western blot analysis of reactivity ofanti-p53^(R175H) antibodies in the serum of mice immunised with TrxR175Hagainst cell lysates from SKBR3 cells. Serum was used at a 1:1000dilution. Lanes A-E contain serum from five different mice, and lane Fis a positive control containing antibody D01. The results show thatafter the second injection all of the mice analysed contained antibodyspecific for full-length R175H mutant p53 protein.

FIG. 74F shows the results of the analysis of the levels of cells ofdifferent immune cell subsets following immunisation with Trx, TrxR175Hor PBS. 3 days after the 5^(th) injection mice were sacrificed andsplenocytes were analysed by flow cytometry. The percentage of T cellswas found to be increased by immunisation with TrxR175H or Trx ascompared to PBS in p53^(R172H/R172H) mutant mice.

Taken together, the ELISA, cell staining, western blot and flowcytometry data demonstrate that TrxR175H can effectively elicit both Tcell and B cell responses to mutant R175H p53.

The invention claimed is:
 1. A method for producing an antibody which isspecific for a mutant p53 polypeptide over wildtype p53 polypeptide,comprising administering to a subject an immunogen, wherein theimmunogen is a peptide or polypeptide comprising: (i) an antigensequence, comprising at least two amino acid sequences of the mutant p53polypeptide including the mutation and at least one amino acidimmediately adjacent to the mutation, wherein the at least two aminoacid sequences of the mutant p53 polypeptide are non-identical; and (ii)a scaffold sequence derived from a thioredoxin for providing the antigensequence in a solvent-accessible configuration.
 2. The method accordingto claim 1, wherein the antigen sequence is inserted in, or substitutedfor all or part of, the active site sequence of the thioredoxin.
 3. Themethod according to claim 1, wherein the peptide or polypeptide used asan immunogen additionally comprises one or more linker sequences betweenthe antigen sequence and the scaffold sequence.
 4. The method accordingto claim 1, wherein the peptide or polypeptide used as an immunogenadditionally comprises linker sequences between the at least two aminoacid sequences of the mutant p53 polypeptide.
 5. The method according toclaim 1, wherein the amino acid sequence of the mutant p53 polypeptidecomprises at least 5 amino acids.
 6. The method according to claim 1,wherein the mutant p53 polypeptide comprises a mutation in theDNA-binding domain (DBD).
 7. The method according to claim 1, whereinthe mutant p53 polypeptide comprises a mutation selected from one ofR175H, R248Q, R273H, R248W, G245S, R273C, R282W, R249S, G245D, C176F,H179Y, H179R, Y220C and R337H.
 8. The method according to claim 1,wherein the mutant p53 polypeptide comprises, or consists of, the aminoacid sequence of one of SEQ ID NOs: 3 to
 16. 9. A cancer vaccine,comprising an immunogen, wherein the immunogen is a peptide orpolypeptide comprising: (i) an antigen sequence, comprising at least twoamino acid sequences of a mutant p53 polypeptide including the mutationand at least one amino acid immediately adjacent to the mutation,wherein the at least two amino acid sequences of the mutant p53polypeptide are non-identical; and (ii) a scaffold sequence derived froma thioredoxin for providing the antigen sequence in a solvent-accessibleconfiguration.
 10. The cancer vaccine of claim 9, wherein the cancer isa cancer comprising a cell or cells expressing a mutant p53 polypeptide,or comprising nucleic acid encoding a mutant p53 polypeptide.
 11. Amethod for vaccinating a subject against a cancer, comprisingadministering to the subject the cancer vaccine of claim 9, therebyvaccinating the subject against a cancer.
 12. The method of claim 11,wherein the cancer is a cancer comprising a cell or cells expressing amutant p53 polypeptide, or comprising nucleic acid encoding a mutantp53.